Opsin polypeptides and methods of use thereof

ABSTRACT

The present disclosure provides opsins, including variant opsins with increased activity and/or increased trafficking to the plasma membrane. The opsins are useful in therapeutic and screening applications, which are also provided.

CROSS-REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 15/334,007, filed Oct. 25, 2016, which is a divisionalapplication of U.S. patent application Ser. No. 15/097,925, filed Apr.13, 2016, now U.S. Pat. No. 9,505,817, which is a divisional applicationof U.S. patent application Ser. No. 14/365,477, filed Jun. 13, 2014, nowU.S. Pat. No. 9,365,628, which is a national stage filing under 35U.S.C. § 371 of PCT/US2012/069133, filed Dec. 12, 2012, which claims thebenefit of U.S. Provisional Patent Application No. 61/576,858, filedDec. 16, 2011, each of which applications is incorporated herein byreference in its entirety.

BACKGROUND

Diverse and elegant mechanisms have evolved to enable organisms toharvest light for a variety of survival functions, including energygeneration and the identification of suitable survival environments. Amajor class of light-sensitive protein consists of 7-transmembranerhodopsins that can be found across all kingdoms of life and serve adiverse range of functions. Many prokaryotes employ these proteins tocontrol proton gradients and to maintain membrane potential and ionichomeostasis, and many motile microorganisms have evolved opsin-basedphotoreceptors to modulate flagellar beating and thereby directphototaxis toward environments with optimal light intensities forphotosynthesis.

Owing to their structural simplicity (both light sensation and effectordomains are encoded within a single gene) and fast kinetics, microbialopsins can be treated as precise and modular photosensitizationcomponents for introduction into non-light sensitive cells to enablerapid optical control of specific cellular processes. In recent years,the development of cellular perturbation tools based on these and otherlight sensitive proteins has resulted in a technology calledoptogenetics, referring to the integration of genetic and opticalcontrol to achieve gain- or loss-of-function of precisely defined eventswithin specified cells of living tissue.

There is a need in the art for depolarizing and hyperpolarizingoptogenetic tools, e.g., for use in controlling neural activity.

SUMMARY

The present disclosure provides opsins, including variant opsins withincreased activity and/or increased trafficking to the plasma membrane.The opsins are useful in therapeutic and screening applications, whichare also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict properties of hyperpolarizing tools.

FIGS. 2A-2F depict performance of hyperpolarizing tools.

FIGS. 3A-3F depict characterization of a ChR from Dunaliella salina. ForFIG. 3B: DChR1 (SEQ ID NO: 15), CChR1 (SEQ ID NO: 16), CChR2 (SEQ ID NO:17), VChR1 (SEQ ID NO: 18), and VChR2 (SEQ ID NO: 19).

FIG. 4 depicts a nucleotide sequence encoding a ChR from Dunaliellasalina (SEQ ID NO: 20).

FIG. 5 depicts a nucleotide sequence encoding a ChR from Dunaliellasalina, codon optimized for expression in mammalian cells (SEQ ID NO:21).

FIG. 6 depicts an amino acid sequence of Dunaliella salina ChR (SEQ IDNO: 22).

FIGS. 7A-7E depict the amino acid sequences of exemplary variant opsins:FIG. 7A (SEQ ID NO: 23); FIG. 7B (SEQ ID NO: 24); FIG. 7C (SEQ ID NO:25); FIG. 7D (SEQ ID NO: 26); and FIG. 7E (SEQ ID NO: 27).

FIGS. 8A-8C depict an amino acid sequence of Halorubrum sodomensearchaerhodopsin-3; and nucleotide sequences encoding same: FIG. 8A (SEQID NO: 28); FIG. 8B (SEQ ID NO: 29); and FIG. 8C (SEQ ID NO: 30).

FIGS. 9A and 9B depict an amino acid sequence of Halorubrum sodomensestrain TP009 opsin; and a nucleotide sequence encoding same: FIG. 9A(SEQ ID NO: 31) and FIG. 9B (SEQ ID NO: 32).

FIGS. 10A-10C depict an amino acid sequence of Leptosphaeria maculansopsin and nucleotides sequences encoding same: FIG. 10A (SEQ ID NO: 33);FIG. 10B (SEQ ID NO: 34); and FIG. 10C (SEQ ID NO: 35).

DEFINITIONS

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxyl grouppresent at the carboxyl terminus of a polypeptide. In keeping withstandard polypeptide nomenclature, J. Biol. Chem., 243 (1969), 3552-59is used.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxynucleotides. Thus, this term includes, but isnot limited to, single-, double-, or multi-stranded DNA or RNA, genomicDNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”). Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid by endonucleases, in a form not normally found in nature. Thus anisolated nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

Nucleic acid sequence identity (as well as amino acid sequence identity)is calculated based on a reference sequence, which may be a subset of alarger sequence, such as a conserved motif, coding region, flankingregion, etc. A reference sequence will usually be at least about 18residues long, more usually at least about 30 residues long, and mayextend to the complete sequence that is being compared. Algorithms forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990), J. Mol. Biol. 215:403-10 (using defaultsettings, i.e. parameters w=4 and T=17).

The term “genetic modification” and refers to a permanent or transientgenetic change induced in a cell following introduction into the cell ofnew nucleic acid (i.e., nucleic acid exogenous to the cell). Geneticchange (“modification”) can be accomplished by incorporation of the newnucleic acid into the genome of the host cell, or by transient or stablemaintenance of the new nucleic acid as an extrachromosomal element.Where the cell is a eukaryotic cell, a permanent genetic change can beachieved by introduction of the nucleic acid into the genome of thecell. Suitable methods of genetic modification include viral infection,transfection, conjugation, protoplast fusion, electroporation, particlegun technology, calcium phosphate precipitation, direct microinjection,and the like.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, or a cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, or thecell naturally occurs. An isolated genetically modified host cell may bepresent in a mixed population of genetically modified host cells. Anisolated polypeptide will in some embodiments be synthetic. “Syntheticpolypeptides” are assembled from amino acids, and are chemicallysynthesized in vitro, e.g., cell-free chemical synthesis, usingprocedures known to those skilled in the art.

By “purified” is meant a compound of interest (e.g., a polypeptide) hasbeen separated from components that accompany it in nature. “Purified”can also be used to refer to a compound of interest separated fromcomponents that can accompany it during manufacture (e.g., in chemicalsynthesis). In some embodiments, a compound is substantially pure whenit is at least 50% to 60%, by weight, free from organic molecules withwhich it is naturally associated or with which it is associated duringmanufacture. In some embodiments, the preparation is at least 75%, atleast 90%, at least 95%, or at least 99%, by weight, of the compound ofinterest. A substantially pure polypeptide can be obtained, for example,by chemically synthesizing the polypeptide, or by a combination ofpurification and chemical modification. A substantially pure polypeptidecan also be obtained by, for example, affinity chromatography. Puritycan be measured by any appropriate method, e.g., chromatography, massspectroscopy, high performance liquid chromatography analysis, etc.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. Insome embodiments, the individual is a human. In some embodiments, theindividual is a murine.

The terms “treatment,” “treating,” “treat,” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

A “therapeutically effective amount” or “efficacious amount” means theamount of an agent that, when administered to a mammal or other subjectfor treating a disease, is sufficient to effect such treatment for thedisease. The “therapeutically effective amount” will vary depending onagent, the disease or condition and its severity and the age, weight,etc., of the subject to be treated.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “avariant opsin polypeptide” includes a plurality of such polypeptides andreference to “the trafficking signal” includes reference to one or moretrafficking signals and equivalents thereof known to those skilled inthe art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides opsins, including variant opsins withincreased activity and/or increased trafficking to the plasma membrane.The opsins are useful in therapeutic and screening applications, whichare also provided.

Opsins

The present disclosure provides opsin polypeptides, and nucleic acids(“opsin nucleic acids”) comprising nucleotide sequences encoding theopsin polypeptides. The present disclosure also provides geneticallymodified host cells comprising an opsin nucleic acid. An opsinpolypeptide is also referred to herein as a “tool.”

A subject isolated opsin polypeptide comprises an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 97%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 500 aminoacids to about 550 amino acids, from about 550 amino acids to about 600amino acids, from about 600 amino acids to about 650 amino acids, fromabout 650 amino acids to about 700 amino acids, or from about 700 aminoacids to 720 amino acids, of the amino acid sequence depicted in FIG. 6.Such an opsin can be referred to as “DChR1.”

A subject isolated opsin polypeptide can have a length of from about 500amino acids to about 550 amino acids, from about 550 amino acids toabout 600 amino acids, from about 600 amino acids to about 650 aminoacids, from about 650 amino acids to about 700 amino acids, or fromabout 700 amino acids to 720 amino acids.

An isolated opsin polypeptide of the present disclosure can be encodedby a nucleotide sequence having at least about 85%, at least about 90%,at least about 95%, at least about 97%, at least about 98%, at leastabout 99%, or 100%, nucleotide sequence identity to a contiguous stretchof from about 1800 nucleotides to about 1900 nucleotides, from about1900 nucleotides to about 2000 nucleotides, from about 2000 nucleotidesto about 2100 nucleotides, or from about 2100 nucleotides to 2163nucleotides, of the nucleotide sequence depicted in FIG. 4 or FIG. 5.

An isolated opsin polypeptide of the present disclosure functions as alight-activated proton channel, e.g., a subject isolated opsin functionsas a proton pump.

In some embodiments, a subject DChR1 opsin is modified to include an ERexport sequence and/or a trafficking sequence, as described in detailbelow. Thus, in some embodiments, a subject DChR1 opsin comprises, inorder from amino terminus to carboxyl terminus, a DChR1 opsin; and an ERexport sequence. In some embodiments, a subject DChR1 opsin comprises,in order from amino terminus to carboxyl terminus, a DChR1 opsin; atrafficking sequence; and an ER export sequence. In some embodiments, asubject DChR1 opsin comprises, in order from amino terminus to carboxylterminus, a DChR1 opsin; a trafficking sequence; an interveningsequence; and an ER export sequence. Suitable ER export sequences,trafficking sequences, and intervening sequences are described in detailbelow.

The present disclosure provides a composition comprising a subject opsinpolypeptide. A subject opsin polypeptide composition can comprise, inaddition to a subject opsin polypeptide, one or more of: a salt, e.g.,NaCl, MgCl₂, KC1, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

Nucleic Acids

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a subject opsin. A nucleotide sequence encoding asubject opsin can be operably linked to one or more regulatory elements,such as a promoter and enhancer, that allow expression of the nucleotidesequence in the intended target cells (e.g., a cell that is geneticallymodified to synthesize the encoded opsin).

In some embodiments, a DChR1-encoding nucleotide sequence has at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%, nucleotide sequenceidentity to a contiguous stretch of from about 1800 nucleotides to about1900 nucleotides, from about 1900 nucleotides to about 2000 nucleotides,from about 2000 nucleotides to about 2100 nucleotides, or from about2100 nucleotides to 2163 nucleotides, of the nucleotide sequencedepicted in FIG. 4. In some cases, the nucleotide sequence iscodon-optimized for expression in a mammalian cell.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LaI repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject opsin can be present in anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding a protein of interest (e.g., an opsin). A selectablemarker operative in the expression host may be present. Suitableexpression vectors include, but are not limited to, viral vectors (e.g.viral vectors based on vaccinia virus; poliovirus; adenovirus (see,e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras etal., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704,1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655);adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., InvestOpthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al.,Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiencyvirus, myeloproliferative sarcoma virus, and mammary tumor virus); andthe like.

Also provided herein is a recombinant vector comprising a subjectpolynucleotide encoding a subject opsin or any variant thereof. Asubject recombinant vector also include vectors comprising apolynucleotide which encodes an RNA (e.g., an mRNA) that whentranscribed from the polynucleotides of the vector will result in theaccumulation of a subject opsin on the plasma membranes of target animalcells. Vectors which may be used include, without limitation,lentiviral, HSV, adenoviral, and andeno-associated viral (AAV) vectors.Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV andEIAV. Lentiviruses may be pseudotyped with the envelope proteins ofother viruses, including, but not limited to VSV, rabies, Mo-MLV,baculovirus and Ebola. Such vectors may be prepared using standardmethods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectorsare DNA viruses of relatively small size that can integrate, in a stableand sites-pecific manner, into the genome of the cells that they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. It encompasses approximately 4700bases and contains an inverted terminal repeat (ITR) region ofapproximately 145 bases at each end, which serves as an origin ofreplication for the virus. The remainder of the genome is divided intotwo essential regions that carry the encapsidation functions: theleft-hand part of the genome, that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (see, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” in Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p5-14,Hudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006),the disclosures of which are hereby incorporated by reference herein intheir entireties). Methods for purifying for vectors may be found in,for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 andInternational Patent Application Publication No.: WO/1999/011764 titled“Methods for Generating High Titer Helper-free Preparation ofRecombinant AAV Vectors”, the disclosures of which are hereinincorporated by reference in their entirety. Preparation of hybridvectors is described in, for example, PCT Application No.PCT/US2005/027091, the disclosure of which is herein incorporated byreference in its entirety. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (See e.g.,International Patent Application Publication Nos: WO 91/18088 and WO93/09239; U.S. Pat. Nos: 4,797,368, 6,596,535, and 5,139,941; andEuropean Patent No: 0488528, all of which are herein incorporated byreference in their entirety). These publications describe variousAAV-derived constructs in which the rep and/or cap genes are deleted andreplaced by a gene of interest, and the use of these constructs fortransferring the gene of interest in vitro (into cultured cells) or invivo (directly into an organism). A replication defective recombinantAAV can be prepared by co-transfecting a plasmid containing the nucleicacid sequence of interest flanked by two AAV inverted terminal repeat(ITR) regions, and a plasmid carrying the AAV encapsidation genes (repand cap genes), into a cell line that is infected with a human helpervirus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

In some embodiments, a subject recombinant vector is encapsidated into avirus particle (e.g. AAV virus particle including, but not limited to,AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the presentdisclosure includes a recombinant virus particle (recombinant because itcontains a recombinant polynucleotide) comprising any of the vectorsdescribed herein. Methods of producing such particles are known in theart and are described in U.S. Pat. No. 6,596,535.

In some cases, a subject opsin nucleic acid comprises a nucleotidesequence encoding the opsin, where the nucleotide sequence is operablylinked to a neuron-specific transcription control element.

Neuron-specific promoters and other control elements (e.g., enhancers)are known in the art. Suitable neuron-specific control sequencesinclude, but are not limited to, a neuron-specific enolase (NSE)promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. Nos.6,649,811, 5,387,742); an aromatic amino acid decarboxylase (AADC)promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147);a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn etal. (2010) Nat. Med. 16:1161); a serotonin receptor promoter (see, e.g.,GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl.Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991)); a GnRHpromoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al.,Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al.,Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter(see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basicprotein (MBP) promoter; a CMV enhancer/platelet-derived growth factor-βpromoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60); a motorneuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No. 7,632,679;and Lee et al. (2004) Development 131:3295-3306); and an alpha subunitof Ca(²⁺)-calmodulin-dependent protein kinase II (CaMKIIα) promoter(see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250).

Host Cells

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce an opsin of the present disclosure.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method.

Suitable mammalian cells include primary cells and immortalized celllines. In some cases, the mammalian cell is a neuron, e.g., anon-immortalized (primary) neuron. In other cases, the mammalian cell isan immortalized cell line.

Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

In some embodiments, the cell is a neuronal cell or a neuronal-likecell. The cells can be of human, non-human primate, mouse, or ratorigin, or derived from a mammal other than a human, non-human primate,rat, or mouse. Suitable cell lines include, but are not limited to, ahuman glioma cell line, e.g., SVGp12 (ATCC CRL-8621), CCF-STTG1 (ATCCCRL-1718), SW 1088 (ATCC HTB-12), SW 1783 (ATCC HTB-13), LLN-18 (ATCCCRL-2610), LNZTA3WT4 (ATCC CRL-11543), LNZTA3WT11 (ATCC CRL-11544),U-138 MG (ATCC HTB-16), U-87 MG (ATCC HTB-14), H4 (ATCC HTB-148), andLN-229 (ATCC CRL-2611); a human medulloblastoma-derived cell line, e.g.,D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185); ahuman tumor-derived neuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060),SK-N-DZ (ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCCCRL-2142), IMR-32 (ATCC CCL-127), etc.; a mouse neuronal cell line,e.g., BC3H1 (ATCC CRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCCCRL-2534), C8-S (ATCC CRL-2535), Neuro-2a (ATCC CCL-131), NB41A3 (ATCCCCL-147), SW10 (ATCC CRL-2766), NG108-15 (ATCC HB-12317); a rat neuronalcell line, e.g., PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC CRL-2006), C6(ATCC CCL-107), F98 (ATCC CRL-2397), RG2 (ATCC CRL-2433), B35 (ATCCCRL-2754), R3 (ATCC CRL-2764), SCP (ATCC CRL-1700), OA1 (ATCC CRL-6538).

Suitable yeast cells include, but are not limited to, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the present invention include, but are not limitedto, Salmonella typhi and S. typhimurium. Suitable Shigella strainsinclude, but are not limited to, Shigella flexneri, Shigella sonnei, andShigella disenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides,Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and thelike. In some embodiments, the host cell is Escherichia coli.

Variant Opsins with Enhanced Membrane Trafficking

The present disclosure provides variant opsins with improved membranetrafficking properties. The present disclosure also provides nucleicacids encoding the variant opsins. In particular, a subject variantopsin is a hyperpolarizing opsin that includes an endoplasmic reticulum(ER) export sequence, a trafficking sequence (TS), or both an ER exportsequence and a TS. The presence of the ER export sequence and/or the TSprovides for enhanced membrane (e.g., plasma membrane) localization andER export. In some cases, a subject variant opsin comprises one or moreadditional amino acids, which may be disposed between the TS and the ERand/or between the opsin and the TS.

Thus, in some cases, a variant opsin comprises, in order from aminoterminus to carboxyl terminus: an opsin polypeptide; a traffickingsequence; and an ER export sequence.

Hyperpolarizing Opsins

Opsin amino acid sequences that are suitable for inclusion in a subjectvariant opsin include, e.g., an amino acid sequence having at leastabout 85%, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity to a contiguous stretchof from about 200 amino acids to about 220 amino acids, from about 220amino acids to about 230 amino acids, from about 230 amino acids toabout 240 amino acids, or from about 240 amino acids to 257 amino acids,of the amino acid sequence depicted in FIG. 8A (Halorubrum sodomensearchaerhodopsin-3).

Opsin amino acid sequences that are suitable for inclusion in a subjectvariant opsin include, e.g., an amino acid sequence having at leastabout 85%, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity to a contiguous stretchof from about 200 amino acids to about 220 amino acids, from about 220amino acids to about 230 amino acids, from about 230 amino acids toabout 240 amino acids, or from about 240 amino acids to 257 amino acids,of the amino acid sequence depicted in FIG. 9A (Halorubrum sodomensestrain TP009 archaerhodopsin).

Opsin amino acid sequences that are suitable for inclusion in a subjectvariant opsin include, e.g., an amino acid sequence having at leastabout 85%, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity to a contiguous stretchof from about 200 amino acids to about 225 amino acids, from about 225amino acids to about 250 amino acids, from about 250 amino acids toabout 275 amino acids, from about 275 amino acids to about 300 aminoacids, or from about 300 amino acids to 313 amino acids, of the aminoacid sequence depicted in FIG. 10A (Leptosphaeria maculans opsin).

Endoplasmic Reticulum Export Sequences

Endoplasmic reticulum (ER) export sequences that are suitable for use ina modified opsin of the present disclosure include, e.g., VXXSL (where Xis any amino acid) (e.g., VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO: 2);etc.); NANSFCYENEVALTSK (SEQ ID NO: 3); FXYENE (SEQ ID NO: 4) (where Xis any amino acid), e.g., FCYENEV (SEQ ID NO: 5); and the like. An ERexport sequence can have a length of from about 5 amino acids to about25 amino acids, e.g., from about 5 amino acids to about 10 amino acids,from about 10 amino acids to about 15 amino acids, from about 15 aminoacids to about 20 amino acids, or from about 20 amino acids to about 25amino acids.

Trafficking Sequences

Trafficking sequences that are suitable for use in a modified opsin ofthe present disclosure comprise an amino acid sequence having 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequenceidentity to an amino acid sequence such as one of the following:

1) the signal peptide of hChR2

(e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 6))

2) the β2 subunit signal peptide of the neuronal nicotinic acetylcholinereceptor

(e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO: 7));

3) a nicotinic acetylcholine receptor signal sequence

(e.g., MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO: 8));

4) a nicotinic acetylcholine receptor signal sequence

(e.g., MRGTPLLLVVSLFSLLQD (SEQ ID NO: 9));

5) a signal sequence of human inward rectifier potassium channel Kir2.1

(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10)).

A trafficking sequence can have a length of from about 10 amino acids toabout 50 amino acids, e.g., from about 10 amino acids to about 20 aminoacids, from about 20 amino acids to about 30 amino acids, from about 30amino acids to about 40 amino acids, or from about 40 amino acids toabout 50 amino acids.

Additional Sequences

As noted above, in some embodiments, a subject variant opsin comprisesone or more amino acids in addition to the opsin, the TS, and the ERexport sequence. For example, in some embodiments, a subject variantopsin comprises, in order from amino terminus to carboxyl terminus: anopsin; a TS; an intervening amino acid sequence; and an ER export signalsequence.

Suitable intervening amino acid sequences include, e.g., linkers;epitope tags; fluorescent proteins; peptides that provide for ease ofpurification; cleavable linker peptides; and the like.

Suitable fluorescent proteins that can be included in a subject variantopsin include, but are not limited to, a green fluorescent protein fromAequoria victoria or a mutant or derivative thereof e.g., as describedin U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, manysuch GFP which are available commercially, e.g., from Clontech, Inc.; ared fluorescent protein; a yellow fluorescent protein; mCherry; any of avariety of fluorescent and colored proteins from Anthozoan species, asdescribed in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973;and the like.

Exemplary Variant Opsins

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 8A (Halorubrum sodomense archaerhodopsin-3);and b) an ER export sequence. For example, the ER export sequence isselected from VXXSL (where X is any amino acid) (e.g., VKESL (SEQ IDNO:1); VLGSL (SEQ ID NO:2);VLGSL (SEQ ID NO:2); etc.); NANSFCYENEVALTSK(SEQ ID NO:3); and FXYENE (SEQ ID NO:4) (where X is any amino acid),e.g., FCYENEV (SEQ ID NO:5).

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 8A (Halorubrum sodomense archaerhodopsin-3);b) a fluorescent protein; and c) an ER export sequence. For example, theER export sequence is selected from VXXSL (where X is any amino acid)(e.g., VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO:2); VLGSL (SEQ ID NO:2);etc.); NANSFCYENEVALTSK (SEQ ID NO:3); and FXYENE (SEQ ID NO:4) (where Xis any amino acid), e.g., FCYENEV (SEQ ID NO:5).

In some embodiments, a subject variant opsin comprises an amino acidsequence having at least about 85%, or at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identityto the amino acid sequence depicted in FIG. 7A.

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 8A (Halorubrum sodomense archaerhodopsin-3);b) a TS sequence; and c) an ER export sequence. For example, a TSsequence can comprise an amino acid sequence having 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity toan amino acid sequence selected from: MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:6); MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:7);MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:8); MRGTPLLLVVSLFSLLQD (SEQ ID NO:9); and KSRITSEGEYIPLDQIDINV (SEQ ID NO:10). For example, the ER exportsequence is selected from VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:1); VLGSL (SEQ ID NO:2); etc.); NANSFCYENEVALTSK (SEQ IDNO:3); and FXYENE (SEQ ID NO:4) (where X is any amino acid), e.g.,FCYENEV (SEQ ID NO:5).

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 8A (Halorubrum sodomense archaerhodopsin-3);b) a TS sequence; c) a fluorescent protein; and d) an ER exportsequence. For example, a TS sequence can comprise an amino acid sequencehaving 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence selected from:MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO:6); MAGHSNSMALFSFSLLWLCSGVLGTEF(SEQ ID NO:7); MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:8); MRGTPLLLVVSLFSLLQD(SEQ ID NO:9); and KSRITSEGEYIPLDQIDINV (SEQ ID NO:10). For example, theER export sequence is selected from VXXSL (where X is any amino acid)(e.g., VKESL (SEQ ID NO:1); VLGSL (SEQ ID NO:2); etc.); NANSFCYENEVALTSK(SEQ IDNO:3); and FXYENE (SEQ ID NO:4) (where X is any amino acid),e.g., FCYENEV (SEQ ID NO:5).

In some embodiments, a subject variant opsin comprises an amino acidsequence having at least about 85%, or at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identityto the amino acid sequence depicted in FIG. 7B.

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 9A (Halorubrum sodomense strain TP009 opsin);b) a TS sequence; and c) an ER export sequence. For example, a TSsequence can comprise an amino acid sequence having 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity toan amino acid sequence selected from: MDYGGALSAVGRELLFVTNPVVVNGS (SEQ IDNO:6); MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:7);MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:8); MRGTPLLLVVSLFSLLQD (SEQ ID NO:9);and KSRITSEGEYIPLDQIDINV (SEQ ID NO:10). For example, the ER exportsequence is selected from VXXSL (where X is any amino acid) (e.g., VKESL(SEQ ID NO:1); VLGSL (SEQ ID NO:2); etc.); NANSFCYENEVALTSK (SEQ IDNO:3); and FXYENE (SEQ ID NO:4) (where X is any amino acid), e.g.,FCYENEV (SEQ ID NO:5).

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 220 amino acids, from about 220 amino acids to about 230amino acids, from about 230 amino acids to about 240 amino acids, orfrom about 240 amino acids to 257 amino acids, of the amino acidsequence depicted in FIG. 9A (Halorubrum sodomense strain TP009 opsin);b) a TS sequence; c) a fluorescent protein; and d) an ER exportsequence. For example, a TS sequence can comprise an amino acid sequencehaving 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to an amino acid sequence selected from:MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 6); MAGHSNSMALFSFSLLWLCSGVLGTEF(SEQ ID NO: 7); MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO: 8);MRGTPLLLVVSLFSLLQD (SEQ ID NO: 9); and KSRITSEGEYIPLDQIDINV (SEQ ID NO:10). For example, the ER export sequence is selected from VXXSL (where Xis any amino acid) (e.g., VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO: 2);etc.); NANSFCYENEVALTSK (SEQ ID NO: 3); and FXYENE (SEQ ID NO: 4) (whereX is any amino acid), e.g., FCYENEV (SEQ ID NO: 5).

In some embodiments, a subject variant opsin comprises an amino acidsequence having at least about 85%, or at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identityto the amino acid sequence depicted in FIG. 7C.

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 225 amino acids, from about 225 amino acids to about 250amino acids, from about 250 amino acids to about 275 amino acids, fromabout 275 amino acids to about 300 amino acids, or from about 300 aminoacids to 313 amino acids, of the amino acid sequence depicted in FIG.10A (Leptosphaeria maculans opsin); and b) an ER export sequence. Forexample, the ER export sequence is selected from VXXSL (where X is anyamino acid) (e.g., VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO: 2); etc.);NANSFCYENEVALTSK (SEQ ID NO: 3); and FXYENE (SEQ ID NO: 4) (where X isany amino acid), e.g., FCYENEV (SEQ ID NO: 5).

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 225 amino acids, from about 225 amino acids to about 250amino acids, from about 250 amino acids to about 275 amino acids, fromabout 275 amino acids to about 300 amino acids, or from about 300 aminoacids to 313 amino acids, of the amino acid sequence depicted in FIG.10A (Leptosphaeria maculans opsin); b) a fluorescent protein; and c) anER export sequence. For example, the ER export sequence is selected fromVXXSL (where X is any amino acid) (e.g., VKESL (SEQ ID NO: 1); VLGSL(SEQ ID NO: 2); etc.); NANSFCYENEVALTSK (SEQ ID NO: 3); and FXYENE (SEQID NO: 4) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 5).

In some embodiments, a subject variant opsin comprises an amino acidsequence having at least about 85%, or at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identityto the amino acid sequence depicted in FIG. 7D.

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 225 amino acids, from about 225 amino acids to about 250amino acids, from about 250 amino acids to about 275 amino acids, fromabout 275 amino acids to about 300 amino acids, or from about 300 aminoacids to 313 amino acids, of the amino acid sequence depicted in FIG.10A (Leptosphaeria maculans opsin); b) a TS sequence; and c) an ERexport sequence. For example, a TS sequence can comprise an amino acidsequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, amino acid sequence identity to an amino acid sequence selectedfrom:

(SEQ ID NO: 6) MDYGGALSAVGRELLFVTNPVVVNGS; (SEQ ID NO: 7)MAGHSNSMALFSFSLLWLCSGVLGTEF; (SEQ ID NO: 8) MGLRALMLWLLAAAGLVRESLQG;(SEQ ID NO: 9) MRGTPLLLVVSLFSLLQD; and (SEQ ID NO: 10)KSRITSEGEYIPLDQIDINV. For example, the ER export sequence is selectedfrom VXXSL (where X is any amino acid) (e.g.,VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO: 2); etc.);NANSFCYENEVALTSK (SEQ ID NO: 3); and FXYENE (SEQID NO: 4) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 5).

In some embodiments, a subject variant opsin comprises, in order fromamino terminus to carboxyl terminus: a) a hyperpolarizing opsincomprising an amino acid sequence having at least about 85%, or at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 200 aminoacids to about 225 amino acids, from about 225 amino acids to about 250amino acids, from about 250 amino acids to about 275 amino acids, fromabout 275 amino acids to about 300 amino acids, or from about 300 aminoacids to 313 amino acids, of the amino acid sequence depicted in FIG.10A (Leptosphaeria maculans opsin); b) a TS sequence; c) a fluorescentprotein; and d) an ER export sequence. For example, a TS sequence cancomprise an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to an aminoacid sequence selected from:

(SEQ ID NO: 6) MDYGGALSAVGRELLFVTNPVVVNGS; (SEQ ID NO: 7)MAGHSNSMALFSFSLLWLCSGVLGTEF; (SEQ ID NO: 8) MGLRALMLWLLAAAGLVRESLQG;(SEQ ID NO: 9) MRGTPLLLVVSLFSLLQD; and (SEQ ID NO: 10)KSRITSEGEYIPLDQIDINV. For example, the ER export sequence is selectedfrom VXXSL (where X is any amino acid) (e.g.,VKESL (SEQ ID NO: 1); VLGSL (SEQ ID NO: 2); etc.);NANSFCYENEVALTSK (SEQ ID NO: 3); and FXYENE (SEQID NO: 4) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 5).

In some embodiments, a subject variant opsin comprises an amino acidsequence having at least about 85%, or at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identityto the amino acid sequence depicted in FIG. 7E.

Nucleic Acids

The present disclosure provides nucleic acids comprising a nucleotidesequence encoding a subject variant opsin. A nucleotide sequenceencoding a subject variant opsin can be operably linked to one or moreregulatory elements, such as a promoter and enhancer, that allowexpression of the nucleotide sequence in the intended target cells(e.g., a cell that is genetically modified to synthesize the encodedvariant opsin). In some cases, the variant opsin-encoding nucleotidesequence is operably linked to a transcriptional control element(s) thatprovides for neuron-specific expression. In some cases, a nucleotidesequence encoding a subject variant opsin is codon-optimized forexpression in a mammalian cell.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25promoter, a METS promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the Lad repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject opsin can be present in anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding a protein of interest (e.g., a variant opsin). Aselectable marker operative in the expression host may be present.Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiencyvirus, myeloproliferative sarcoma virus, and mammary tumor virus); andthe like.

Also provided herein is a recombinant vector comprising a subjectpolynucleotide encoding a subject variant opsin or any variant thereof.A subject recombinant vector also include vectors comprising apolynucleotide which encodes an RNA (e.g., an mRNA) that whentranscribed from the polynucleotides of the vector will result in theaccumulation of a subject opsin on the plasma membranes of target animalcells. Vectors which may be used include, without limitation,lentiviral, HSV, adenoviral, and andeno-associated viral (AAV) vectors.Lentiviruses include, but are not limited to HIV-1, HIV-2, SIV, FIV andEIAV. Lentiviruses may be pseudotyped with the envelope proteins ofother viruses, including, but not limited to VSV, rabies, Mo-MLV,baculovirus and Ebola. Such vectors may be prepared using standardmethods in the art.

In some embodiments, the vector is a recombinant AAV vector. AAV vectorsare

DNA viruses of relatively small size that can integrate, in a stable andsite-specific manner, into the genome of the cells that they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. It encompasses approximately 4700bases and contains an inverted terminal repeat (ITR) region ofapproximately 145 bases at each end, which serves as an origin ofreplication for the virus. The remainder of the genome is divided intotwo essential regions that carry the encapsidation functions: theleft-hand part of the genome, that contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, that contains the cap gene encoding the capsidproteins of the virus.

AAV vectors may be prepared using standard methods in the art.Adeno-associated viruses of any serotype are suitable (see, e.g.,Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R.Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.Tattersall “The Evolution of Parvovirus Taxonomy” in Parvoviruses (J RKerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p5-14,Hudder Arnold, London, UK (2006); and D E Bowles, J E Rabinowitz, R JSamulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R MLinden, C R Parrish, Eds.) p15-23, Hudder Arnold, London, UK (2006), thedisclosures of which are hereby incorporated by reference herein intheir entireties). Methods for purifying for vectors may be found in,for example, U.S. Pat. Nos. 6 566 118, 6 989 264, and 6 995 006 andInternational Patent Application Publication No.: WO/1999/011764 titled“Methods for Generating High Titer Helper-free Preparation ofRecombinant AAV Vectors”, the disclosures of which are hereinincorporated by reference in their entirety. Preparation of hybridvectors is described in, for example, PCT Application No.PCT/US2005/027091, the disclosure of which is herein incorporated byreference in its entirety. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (See e.g.,International Patent Application Publication Nos: WO 91/18088 and WO93/09239; U.S. Pat. Nos: 4,797,368, 6,596,535, and 5,139,941; andEuropean Patent No: 0488528, all of which are herein incorporated byreference in their entirety). These publications describe variousAAV-derived constructs in which the rep and/or cap genes are deleted andreplaced by a gene of interest, and the use of these constructs fortransferring the gene of interest in vitro (into cultured cells) or invivo (directly into an organism). A replication defective recombinantAAV can be prepared by co-transfecting a plasmid containing the nucleicacid sequence of interest flanked by two AAV inverted terminal repeat(ITR) regions, and a plasmid carrying the AAV encapsidation genes (repand cap genes), into a cell line that is infected with a human helpervirus (for example an adenovirus). The AAV recombinants that areproduced are then purified by standard techniques.

In some embodiments, a subject recombinant vector is encapsidated into avirus particle (e.g. AAV virus particle including, but not limited to,AAV1, AAV2, AAV3, AAV4, AAVS,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the presentdisclosure includes a recombinant virus particle (recombinant because itcontains a recombinant polynucleotide) comprising any of the vectorsdescribed herein. Methods of producing such particles are known in theart and are described in U.S. Pat. No. 6,596,535.

As noted above, in some cases, a subject variant opsin-encodingnucleotide sequence is operably linked to a neuron-specific promoter.Neuron-specific promoters and other control elements (e.g., enhancers)are known in the art. Suitable neuron-specific control sequencesinclude, but are not limited to, a neuron-specific enolase (NSE)promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. Nos.6,649,811, 5,387,742); an aromatic amino acid decarboxylase (AADC)promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147);a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1promoter (see, e.g., Chen et al. (1987) Cell 51:7-19); a serotoninreceptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylasepromoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) andNeuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al.,Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see,e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter(see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652(1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J.17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMVenhancer/platelet-derived growth factor-β promoter (see, e.g., Liu etal. (2004) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2004)Development 131:3295-3306); and an alpha subunit ofCa(²⁺)-calmodulin-dependent protein kinase II (CaMKIIα) promoter (see,e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250).

Utility

A subject opsin finds use in modulating the voltage potential of a cell.A subject opsin finds use in therapeutic and drug screeningapplications. A subject opsin finds use in generating disease models.

Modulating the Voltage Potential of a Cell

For example, a subject opsin is useful for modulating the voltagepotential of a cell, e.g., a neuron. The cell can be in vitro or invivo. Thus, e.g., the present disclosure provides a method for adjustingthe voltage potential of cells, subcellular regions, or extracellularregions, the method generally involving: introducing a nucleic acidcomprising a nucleotide sequence encoding a subject opsin (e.g., alight-driven proton pump) into at least one target cell, subcellularregion, or extracellular region, the opsin operating to changetransmembrane potential in response to a specific wavelength of light;and causing the expression of the nucleic acid by exposing the targetcell, subcellular region, or extracellular region to the specificwavelength of light in a manner designed to cause the voltage potentialof the target cell, subcellular region, or extracellular region toincrease or decrease.

In some cases, a subject method further involves the step of increasingor decreasing the voltage potential of the target cell, subcellularregion, or extracellular region until it is hyperpolarized. Where thetarget cell, subcellular region, or extracellular region is a neuron,the hyperpolarization achieves neural silencing.

In some cases, a subject method further involves the step of using aplurality of different opsins (e.g., light-activated proton pumps)responsive to different wavelengths of light to achieve multi-colorneural silencing by the steps of: expressing each opsin (e.g.,light-activated proton pump) in a different population of cells; andilluminating the cells with different colors of light.

The present disclosure provides a method for adjusting the pH of a cell,subcellular region, or extracellular region, the method generallyinvolving: introducing a nucleic acid comprising a nucleotide sequenceencoding a subject opsin (e.g., light-driven proton pump) into at leastone target cell, subcellular region, or extracellular region, the opsinoperating to change cell, subcellular region, or extracellular region pHin response to a specific wavelength of light; and causing theexpression of the nucleic acid by exposing the target cell, subcellularregion, or extracellular region to the specific wavelength of light in amanner designed to cause the pH of the target cell, subcellular region,or extracellular region to increase or decrease.

The present disclosure provides method for causing cells, subcellularregions, or extracellular regions to release protons as chemicaltransmitters, the method generally involving: introducing a nucleic acidcomprising a nucleotide sequence encoding a subject opsin (e.g.,light-driven proton pump) into at least one target cell, subcellularregion, or extracellular region, the opsin operating to cause protonrelease in response to a specific wavelength of light; and causing theexpression of the nucleic acid by exposing the target cell, subcellularregion, or extracellular region to the specific wavelength of light in amanner designed to cause the target cell, subcellular region, orextracellular region to release protons.

Target Cell-Modulating Applications

In some embodiments, a target cell is genetically modified with asubject nucleic acid (e.g., a nucleic acid comprising a nucleotidesequence encoding an opsin, e.g., a variant opsin). In some cases,target cells are neurons located in the brain of a mammal. The targetcells are genetically modified to express a photosensitive opsin, forexample, a subject opsin (e.g., a subject variant opsin), as describedabove. Light can then be used to stimulate the neurons. Depending upon anumber of factors, such as the location within the brain and thefrequency and length of stimulation, different objectives can beachieved. For instance, current techniques for deep brain stimulus (DBS)use electrodes to apply a current directly to the targeted area of thebrain. The frequency of the electrical stimulus is sometimes referred toas either low-frequency DBS or high-frequency DBS. Studies havesuggested that high-frequency DBS inhibits the generation of impulsesfrom the stimulated cells, while low-frequency DBS facilitates thegeneration of impulses from the stimulated cells. The frequencies thatproduce the effects of high-frequency of low-frequency DBS have alsobeen shown to vary depending upon the specific area of the brain beingstimulated. According to one example of high-frequency DBS, the neuronsare stimulated using electrodes supplying current pulses at frequenciesaround 100 Hz or more. Such a frequency has been shown to be effectivein certain applications, as discussed further herein.

A specific example of DBS is used for the treatment of Parkinson'sdisease. In this application, DBS is often applied to the globuspallidus interna, or the subthalamic nucleus within a patient's brain.By implanting a biological arrangement that modifies the cells torespond to light, a light flashing light can be used in place ofelectrodes. Thus, the targeted neuron cells and external electricalsignal need not be directly applied to the targeted cells. Moreover,light can often travel from its point of origin farther thanelectricity, thereby increasing the effective area relative to thestimulation source and only those neurons that have been photosensitizedare stimulated.

As with the electrode-based DBS methods, one embodiment of the presentinvention can be implemented using high-frequency DBS to inhibit neurongenerated impulses. While high-frequency DBS has been accomplished atfrequencies around 100 Hz, high-frequency DBS using various embodimentsof the present disclosure may not necessarily require the samefrequency. For instance, it may be possible to reproduce the inhibitingeffects of high-frequency DBS at lower frequencies (e.g., 50 Hz) whenusing light activated techniques. For example, activation of ahyperpolarizing opsin can result in hyperpolarization and resistance toaction potential generation. Various frequencies can be used dependingupon the particular application (e.g., the targeted portion of the brainand the desired effect), and the stimulation modality being applied.

Consistent with another example embodiment of the present invention,gene transfer vectors inducing the expression of photosensitivebio-molecules are used to target a specific type of cell. For instance,viral-based proteins (e.g., lentiviruses, adeno-associated viruses orretroviruses) can be created to target specific types of cells, basedupon the proteins that they uniquely express. The targeted cells arethen infected by the viral-based gene-transfer proteins, and begin toproduce a new type of ion channel (for example a subject opsin; asubject variant opsin), thereby becoming photosensitive. This can beparticularly useful for stimulating the targeted cells withoutstimulating other cells that are in proximity to the targeted cells. Forexample, neurons of disparate length, diameter, chronaxie, othermembrane properties, electrical insulation, neurotransmitter output, andoverall function, lie in close proximity to one another, and thus, canbe inadvertently stimulated when using electrodes to provide thestimulation of the neurons. See, e.g., Gradinaru et al. (2007) J.Neurosci. 27(52): 14231-14238, Zhang et al. (2007) Nature 446: 633-639,Zhang et al. (2007) Nature Reviews Neuroscience Vol. 8: 577-581.

The present disclosure provides an implantable arrangement for in vivouse. A light-emitting diode, laser or similar light source is includedfor generating light. A biological portion that modifies target cells toinclude light responsive molecules which facilitate stimulation of thetarget cells in response to light generated by the light source.

Another embodiment of the present invention employs an arrangement forstimulating target cells using a photosensitive protein that allows thetarget cells to be stimulated in response to light. A biologicaldelivery device is used for implanting vectors that modify the targetcells to include the photosensitive protein. An implantation component(e.g., an implantable component comprising a recombinant expressionvector encoding a subject opsin), is used for implanting a lightgenerating device near the target cells. A control device is used foractivating the light generating device to generate light to be receivedby the target cells, thereby stimulating the target cells in response tothe generated light.

For example, light can be delivered to a site internal to an organism(e.g., a mammal). A light generator, such as an an implantable devicethat generates light in vivo, is used. A subject opsin (e.g., a subjectvariant opsin) present in target cells at the site provides forstimulation of the target cells in response to light generated by thelight generator, which light strikes the target cells. The lightgenerator can be a small electronic device on the order of a fewmillimeters in size. The small size is particularly useful forminimizing the intrusiveness of the device and associated implantationprocedure. In another instance, the light generator can include a fiberoptic device that can be used to transmit light from an external sourceto the target cells. For example, the target cells are modified tocontain light-activated proton pump/channel proteins.

A subject light-sensitive protein can be implanted using a number ofdifferent methods. Example methods include, but are not limited to, theuse of various delivery devices, such as gelatin capsules, liquidinjections and the like. Such methods also include the use ofstereotactic surgery techniques such as frames or computerized surgicalnavigation systems to implant or otherwise access areas of the body.

As one example, target cells that have been modified to bephotosensitive, e.g., modified to produce a subject opsin (e.g., asubject variant opsin). The target cells are thus photosensitive.Stimulation of the target cells can be controlled by the implantabledevice. For example, a control circuit can be arranged to respond to anexternal signal by activating, or deactivating a light source, or bycharging the battery that powers light source. In one instance, theexternal signal is electromagnetic radiation that is received by acontrol circuit. For example, radio frequency (RP) signals can betransmitted by an external radiofrequency (RF) transmitter and receivedby a control circuit. In another example, a magnetic field can be usedto activate and/or power the control circuit.

A control circuit can be implemented using varying degrees ofcomplexity. In one instance, the circuit is a simple coil that whenexposed to a magnetic field generates a current. The current is thenused to a power light source. Such an implementation can be particularlyuseful for limiting the size and complexity as well as increasing thelongevity of the device. In another instance, a control circuit caninclude an RF antenna. Optionally, a battery or similar power source,such as a capacitive element, can be used by a control circuit. Whilecharged, the power source allows the circuitry to continue to operatewithout need for concurrent energy delivery from outside the body. Thiscan be particularly useful for providing precise control over the lightemitted by a light source and for increased intensity of the emittedlight. In one embodiment, a light source is implemented using alight-emitting-diode (LED). LEDs have been proven to be useful for lowpower applications and also to have a relatively fast response toelectrical signals.

In another embodiment, a matrix (e.g., which can include a gelatin orsimilar substance) that contains recombinant expression vectors encodinga subject opsin (e.g., a subject variant opsin), which recombinantexpression vectors enter target cells and provide for target cellphotosensitivity. In one instance, the vectors are released onceimplanted into the body. This can be accomplished, for example, by usinga containment material that allows the vectors to be released intoaqueous solution {e.g., using dehydrated or water soluble materials suchas gelatins). The release of the vectors results in the target cellsbeing modified such that they are simulated in response to light from alight source.

In another embodiment, a synthetic mesh that contains the photosensitivecells is used. In one instance, the cells are neurons that have beenmodified to be photosensitive (e.g., modified to include a subjectopsin, e.g., a subject variant opsin. The synthetic mesh can beconstructed so as to allow the dendrites and axons to pass through themess without allowing the entire neuron {e.g., the cell body) to pass.One example of such a mesh has pores that are on the order of 3-7microns in diameter and is made from polyethylene terephthalate. Inanother example embodiment, an injection mechanism is used to deliver asubject opsin (e.g., a subject variant opsin), e.g., a recombinantexpression vector encoding a subject opsin.

For example, an implantable device can be responsive to a magneticfield. For example, an inductor generates a current/Voltage in responseto a magnetic field. The current is passed to a control circuit througha conductive path. In response, a control circuit activates a lightsource using a conductive path. A light source illuminates a biologicalportion in order to stimulate the target cells. In one instance, thebiological portion includes a gelatin, synthetic mesh, or injectionmechanism as discussed above.

In one embodiment, the control portion can be a simple electricalconnection, resistive element, or can be removed completely. In such anembodiment, the intensity, duration and frequency of light generatedwould be directly controlled by the current generated from a magneticfield. This can be particularly useful for creating inexpensive, longlasting and small devices.

In another embodiment, the control portion can be implemented as a morecomplex circuit. For instance the control circuit may include andotherwise implement different rectifier circuits, batteries, pulsetimings, comparator circuits and the like. In a particular example, thecontrol circuit includes an integrated circuit (IC) produced using CMOSor other processes. Integrated circuit technology allows for the use ofa large number of circuit elements in a very small area, and thus, arelatively complex control circuit can be implemented for someapplications.

As an example, an inductor is a surface mount inductor, such as a 100 uHinductor part number CF1008-103K supplied by Gowanda Electronics Corp.The light generating portion is a blue LED, such as LEDs in 0603 or 0805package sizes. A particular example is a blue surface mount LED havingpart number SML0805, available from LEDtronics, Inc (Torrance, Calif.).Connective paths can be implemented using various electrical conductors,such as conductive epoxies, tapes, solder or other adhesive materials.LEDs emitting light in the appropriate spectrum (as applicable to asubject opsin) are available through commercial sources including thissame manufacturer.

The present disclosure provides a method for genetically modifyingneurons to express a light-sensitive opsin described herein. Forexample, a subject opsin can be used to impart photosensitivity uponmammalian nerve cells, by using an expression vector to deliver anucleic acid encoding a subject opsin into targeted nerve cells, whichsubsequently produce the encoded opsin. Stimulation of the target cellswith light results in hyperpolarization of the target cells.

The present disclosure provides methods for generating an inhibitoryneuron-current flow in a neuron, the methods involving modifying theneuron to express a subject opsin; and exposing the neuron to a lightstimulus. The present disclosure provides methods for controlling actionpotential of a neuron, the methods involving modifying the neuron toexpress a subject opsin; and exposing the neuron to a light stimulus.The present disclosure provides methods for controlling a voltage levelacross a cell membrane of a cell, the methods involving modifying thecell to express a subject opsin; and exposing the cell to a lightstimulus.

The present disclosure provides a system for controlling an actionpotential of a neuron in vivo. The system includes a delivery device, alight source, and a control device. The delivery device introduces alight responsive protein (a subject opsin) to the neuron, with the lightresponsive protein producing an inhibitory current. The light sourcegenerates light for stimulating the light responsive protein, and thecontrol device controls the generation of light by the light source.

The present disclosure provides methods of treating a disorder. Themethod targets a group of neurons associated with the disorder; thetarget neurons are modified to express a subject opsin; the modifiedtarget neurons produce an inhibitory current that reduces depolarizationof the neurons; the modified neurons are exposed to a light stimulus,thereby reducing depolarization of the neurons.

Drug Screening

Certain embodiments of the present invention can be useful in drugscreening. The various light-sensitive proteins, serving to regulatemembrane voltage using ion switches that, when activated (ordeactivated) in response to light, function as channels or pumps and arereferred to hereafter as light-responsive ion switches orlight-activated membrane potential switches (LAMPS).

For example, the present disclosure provides for screening forion-channel and ion-pump affecting compounds. The system introduces oneor more drug candidates that could either block or enhance the activityof a subject opsin in a cell modified to synthesize a subject opsin.Light triggers optically responsive ion channels in the cells causing achange in the voltage seen across the cell membrane. The voltage changestimulates voltage-gated ion channels in the cells which will then causea change in ion concentrations that can be read as optical outputs.These optical signals are detected and used to determine what effect, ifany, the drug candidates have on the voltage-gated ion channels. In amore specific embodiment a protein expressing a proton pump isintroduced into the cell.

In one instance, the system allows for different drug candidates to bescreened without necessitating extensive setup between screenings. Forexample, an assay may be performed using optics both to stimulate theoptically responsive cells and to detect the effectiveness of the drug.The use of optics instead of manual contacts, e.g., using a whole-cellpatch clamp, can be particularly useful in increasing the throughput ofthe assay screening process. For instance, the time between screeningscan be reduced by minimizing or eliminating physical manipulationsotherwise necessary to stimulate or detect ion flow in the target cells.The cells can also be prepared prior to the screening process becausethe test equipment need only be optically coupled to the prepared cells.In another instance, throughput may be increased by screening a numberof different drugs simultaneously using, for example, an array of photodetectors and a corresponding array of modified cells exposed todifferent drugs.

Optical stimulation of the modified cells can be altered to determinespecific properties of an introduced drug candidate. For example, theintensity of the optical stimulus may be modified to change thecorresponding level of depolarization. The level of desireddepolarization can be tuned to further characterize the effectiveness ofthe drug under test. In another example, the optical stimulus mayinclude rapid pulsing of the light. By correlating the temporalrelationship between the optical stimulus and the resultant detectedfluorescence, the drug may be further characterized in terms of akinetic response. Thus, the drug may be characterized for a variety ofdifferent aspects including, but not limited to, the steady state effecton ion concentrations, a change in the level of depolarization necessaryto open voltage gated ion channels, and the effect on repeateddepolarization.

In one embodiment, the system allows for simple calibration of theoptical stimulation and/or detection. The modified cells may beoptically stimulated prior to introduction of the drug candidate. Theion channel responsiveness is detected and recorded. The recorded valuesmay be used as a baseline for comparison to the ion channelresponsiveness of the same modified cells after the introduction of thedrug under test. The recorded values may also be used to modify theoptical stimulus or the sensitivity of the optical detector. Suchmodifications may be applied to an individual test sample or an array oftest samples. For such an array of test samples, each test sample may beindividually calibrated by adjusting the corresponding optical stimulus.Similarly, each corresponding photo detector may be individuallyadjusted.

The amount of time allotted for light delivery may vary, and depends onfactors including the level of light-gated proton or ion channel/pumpexpression, and the density and characteristics of other proton/ionicchannel characteristics of that cell population. The amount of timeallotted for light receipt may vary, and depends upon factors includingthe degree of accuracy required for the screening session. The amount oftime allotted for well-plate (tray) changing may vary, and depends uponfactors including the mechanical speed of the automated apparatus. Iffast neurons are used as the cells being tested, the cellularstimulation and LEIA detection process may be accomplished inmilliseconds.

The process above may be repeated under varying conditions. For example,a given set of cells may be tested with no drug present, andsubsequently with one or more drugs present. The response ofelectrically-excitable cells under those conditions may be therebydocumented, compared and studied. If the invention is implemented withat least one emitter/detector for each well on a tray and at least twoconcurrently operating devices, continuous operation may be maintainedfor extended periods of time.

Exemplary screening methods could include the collection of multipledata points without having to switch samples. Because control over thesamples is reversible in the same sample preparation by simply turningthe activating light on and off with fast shutters, the same samples canbe reused. Further, a range of patterns of stimulation can be providedto the same cell sample so that testing can be performed for the effectof drugs without concern with regards to differences across differentsample preparations. By modulating the level of excitation (e.g., byramping the level from no light to a high or maximum intensity), theeffect of the drug across a range of membrane potentials can be tested.This permits for the identification of drugs that are efficacious duringhyperpolarized, natural, or depolarized membrane potentials.

The cell lines described herein may be a particularly useful fordetailed characterization of drug candidates in a high-throughputmanner. Optical control is relatively fast, thereby allowing for thetesting the drug's activity under more physiological forms ofactivation. For example, different frequencies of depolarization and/orhyperpolarization may be used to determine how a drug interacts with thechannel under physiological forms of neural activity. In some instances,the process may be accomplished without the application of expensivechemical dyes to the cell lines.

In conjunction with the various properties discussed herein, the use ofvarious embodiments of the invention may be particularly useful forimproving screening throughput by eliminating the need for cumbersomemechanical manipulation and liquid handling. Various embodiments mayalso be useful for repeatable the screening assay using the samesamples, reducing screening cost by eliminating the need forchemically-based fluorescence reports, producing high temporal precisionand low signal artifact (due to the optical nature of the voltagemanipulation), modulating the level of depolarization by attenuating thelight intensity used for stimulation, and ascertaining the kinetics ofthe drug's modulation on the ion channel through the use of pulsed lightpatterns.

The existence of multiple independently controllable excitation proteinsand inhibition proteins opens the door for a variety of applicationsincluding, but not limited to, applications for treatment of a varietyof disorders and the use of a plurality of light-responsive proteinsthat can be selected so as to respond to a plurality of respectiveoptical wavelengths. Although not always expressly stated, inhibitioncan be used in combination with, in addition to, or in place ofexcitation in the applications. The family of single-component proteinshas been shown to respond to multiple wavelengths and intensities oflight. Aspects of the disclosure allow for further mutations and/orsearches for sequences that allow for additional optical wavelengthsand/or individually controllable protein channels. Variations on theoptical stimulus (e.g., a wavelength, intensity or duration profile) canalso be used. For instance, stimulation profiles may exploit overlaps inthe excitation wavelengths of two different ion channel proteins toallow excitation of both proteins at the same time. In one suchinstance, the proteins may have different levels of responsibility.Thus, in a neural application, one set of ion channels may producespiking at a different success percentage relative to a second set ofion channels Similarly, the overlaps in inhibition wavelengths of twodifferent ion channels (or pumps) allows for inhibition of both proteinsat the same time.

Alternatively, multiple light sources may be used allowing forstimulations of the light responsive proteins in the combinationdesired, while leaving other proteins unstimulated.

Therapeutic Applications

The present disclosure provides various therapeutic methods.

Addiction is associated with a variety of brain functions, includingreward and expectation. Additionally, the driving cause of addiction mayvary between individuals. According to one embodiment, addiction, forexample nicotine addiction, may be treated with optogeneticstabilization of small areas on the insula. Optionally, functional brainimaging, for example cued-state PET or fMRI, may be used to locate ahyper metabolic focus in order to determine a precise target spot forthe intervention on the insula surface.

Optogenetic excitation of the nucleus accumbens and septum may providereward and pleasure to a patient without need for resorting to use ofsubstances, and hence may hold a key to addiction treatment. Conversely,optogenetic stabilization of the nucleus accumbens and septum may beused to decrease drug craving in the context of addiction. In analternative embodiment, optogenetic stabilization of hyper metabolicactivity observed at the genu of the anterior cingulate (BA32) can beused to decrease drug craving. Optogenetic stabilization of cells withinthe arcuate nucleus of the medial hypothalamus which contain peptideproducts of pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) can also be used todecrease drug addiction behavior.

Optogenetic stimulation of neuroendocrine neurons of the hypothalamicperiventricular nucleus that secrete somatostatin can be used to inhibitsecretion of growth hormone from the anterior pituitary, for example inacromegaly. Optogenetic stabilization of neuroendocrine neurons thatsecrete somatostatin or growth hormone can be used to increase growthand physical development. Among the changes that accompany “normal”aging, is a sharp decline in serum growth hormone levels after the4^(th) and 5^(th) decades. Consequently, physical deteriorationassociated with aging may be lessened through optogenetic stabilizationof the periventricular nucleus.

Optogenetic stabilization of the ventromedial nucleus of thehypothalamus, particularly the pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) of the arcuatenucleus, can be used to increase appetite, and thereby treat anorexianervosa. Alternatively, optogenetic stimulation of the lateral nuclei ofthe hypothalamus can be used to increase appetite and eating behaviors.

Optogenetic excitation in the cholinergic cells of affected areasincluding the temporal lobe, the NBM (Nucleus basalis of Meynert) andthe posterior cingulate gyrus (BA 31) provides stimulation, and henceneurotrophic drive to deteriorating areas.

Because the affected areas are widespread within the brain, an analogoustreatment with implanted electrodes may be less feasible than anopto-genetic approach.

Anxiety disorders are typically associated with increased activity inthe left temporal and frontal cortex and amygdala, which trends towardnormal as anxiety resolves. Accordingly, the affected left temporal andfrontal regions and amygdala may be treated with optogeneticstabilization, so as to dampen activity in these regions.

In normal physiology, photosensitive neural cells of the retina, whichdepolarize in response to the light that they receive, create a visualmap of the received light pattern. Optogenetic ion channels can be usedto mimic this process in many parts of the body, and the eyes are noexception. In the case of visual impairment or blindness due to damagedretina, a functionally new retina can be grown, which uses naturalambient light rather than flashing light patterns from an implanteddevice. The artificial retina grown may be placed in the location of theoriginal retina (where it can take advantage of the optic nerve servingas a conduit back to the visual cortex). Alternatively, the artificialretina may be placed in another location, such as the forehead, providedthat a conduit for the depolarization signals are transmitted tocortical tissue capable of deciphering the encoded information from theoptogenetic sensor matrix. Cortical blindness could also be treated bysimulating visual pathways downstream of the visual cortex. Thestimulation would be based on visual data produced up stream of thevisual cortex or by an artificial light sensor.

Treatment of tachycardia may be accomplished with optogeneticstimulation to parasympathetic nervous system fibers including CN X orVagus Nerve. This causes a decrease in the S A node rate, therebydecreasing the heart rate and force of contraction. Similarly,optogenetic stabilization of sympathetic nervous system fibers withinspinal nerves T1 through T4, serves to slow the heart. For the treatmentof pathological bradycardia, optogenetic stabilization of the Vagusnerve, or optogenetic stimulation of sympathetic fibers in T1 through T4will serve to increase heart rate. Cardiac disrhythmias resulting fromaberrant electrical foci that outpace the sinoatrial node may besuppressed by treating the aberrant electrical focus with moderateoptogenetic stabilization. This decreases the intrinsic rate of firingwithin the treated tissue, and permits the sinoatrial node to regain itsrole in pacing the heart's electrical system. In a similar way, any typeof cardiac arrhythmia could be treated. Degeneration of cardiac tissuethat occurs in cardiomyopathy or congestive heart failure could also betreated using this invention; the remaining tissue could be excitedusing various embodiments of the invention.

Optogenetic excitation stimulation of brain regions including thefrontal lobe, parietal lobes and hippocampi, may increase processingspeed, improve memory, and stimulate growth and interconnection ofneurons, including spurring development of neural progenitor cells. Asan example, one such application of the present invention is directed tooptogenetic excitation stimulation of targeted neurons in the thalamusfor the purpose of bringing a patient out of a near- vegetative(barely-conscious) state. Growth of light-gated ion channels or pumps inthe membrane of targeted thalamus neurons is affected. These modifiedneurons are then stimulated (e.g., via optics which may also gain accessby the same passageway) by directing a flash of light thereupon so as tomodulate the function of the targeted neurons and/or surrounding cells.

In an alternative embodiment, optogenetic excitation may be used totreat weakened cardiac muscle in conditions such as congestive heartfailure. Electrical assistance to failing heart muscle of CHF isgenerally not practical, due to the thin-stretched, fragile state of thecardiac wall, and the difficulty in providing an evenly distributedelectrical coupling between an electrodes and muscle. For this reason,methods to date for increasing cardiac contractility have involvedeither pharmacological methods such as Beta agonists, and mechanicalapproaches such as ventricular assist devices. In this embodiment of thepresent invention, optogenetic excitation is delivered to weakened heartmuscle via light emitting elements on the inner surface of a jacketsurround the heart or otherwise against the affected heart wall. Lightmay be diffused by means well known in the art, to smoothly cover largeareas of muscle, prompting contraction with each light pulse.

Optogenetic stabilization in the subgenual portion of the cingulate gyms(Cg25), yellow light may be applied with an implanted device. The goalwould be to treat depression by suppressing target activity in manneranalogous to what is taught by Mayberg H S et al, “Deep BrainStimulation for Treatment-Resistant Depression,” Neuron, Vol. 45,651-660, Mar. 3, 2005, pp. 651-660, which is fully incorporated hereinby reference. In an alternative embodiment, an optogenetic excitationstimulation method is to increase activity in that region in a manneranalogous to what is taught by Schlaepfer et al., “Deep Brainstimulation to Reward Circuitry Alleviates Anhedonia in Refractory MajorDepression,” Neuropsychopharmacology 2007, pp. 1-10, which is fullyincorporated herein by reference.

In yet another embodiment, the left dorsolateral prefrontal cortex(LDPFC) is targeted with an optogenetic excitation stimulation method.Pacing the LDLPFC at 5-20 Hz serves to increase the basal metaboliclevel of this structure which, via connecting circuitry, serves todecrease activity in Cg 25, improving depression in the process.Suppression of the right dorsolateral prefrontal cortex (RDLPFC) is alsoan effective depression treatment strategy. This may be accomplished byoptogenetic stabilization on the RDLPFC, or suppression may also beaccomplished by using optogenetic excitation stimulation, and pulsing ata slow rate (e.g. 1 Hz or less) improving depression in the process.Vagus nerve stimulation (VNS) may be improved using an optogeneticapproach. Use of optogenetic excitation may be used in order tostimulate only the vagus afferents to the brain, such as the nodoseganglion and the jugular ganglion.

Efferents from the brain would not receive stimulation by this approach,thus eliminating some of the side-effects of VNS including discomfort inthe throat, a cough, difficulty swallowing and a hoarse voice. In analternative embodiment, the hippocampus may be optogenetically excited,leading to increased dendritic and axonal sprouting, and overall growthof the hippocampus. Other brain regions implicated in depression thatcould be treated using this invention include the amygdala, accumbens,orbitofrontal and orbitomedial cortex, hippocampus, olfactory cortex,and dopaminergic, serotonergic, and noradrenergic projections.Optogenetic approaches could be used to control spread of activitythrough structures like the hippocampus to control depressive symptoms.

So long as there are viable alpha and beta cell populations in thepancreatic islets of Langerhans, the islets can be targeted for thetreatment of diabetes. For example, when serum glucose is high (asdetermined manually or by closed loop glucose detection system),optogenetic excitation may be used to cause insulin release from thebeta cells of the islets of Langerhans in the pancreas, whileoptogenetic stabilization is used to prevent glucagon release from thealpha cells of the islets of Langerhans in the pancreas. Conversely,when blood sugars are too low (as determined manually or by closed loopglucose detection system), optogenetic stabilization may be used to stopbeta cell secretion of insulin, and optogenetic excitation may be usedto increase alpha-cell secretion of glucagon.

For treatment of epilepsy, quenching or blocking epileptogenic activityis amenable to optogenetic approaches. Most epilepsy patients have astereotyped pattern of activity spread resulting from an epileptogenicfocus. Optogenetic stabilization could be used to suppress the abnormalactivity before it spreads or truncated it early in its course.Alternatively, activation of excitatory tissue via optogeneticexcitation stimulation could be delivered in a series of deliberatelyasynchronous patterns to disrupt the emerging seizure activity. Anotheralternative involves the activation of optogenetic excitationstimulation in GABAergic neurons to provide a similar result. Thalamicrelays may be targeted with optogenetic stabilization triggered when anabnormal EEG pattern is detected.

Another embodiment involves the treatment of gastrointestinal disorders.The digestive system has its own, semi-autonomous nervous systemcontaining sensory neurons, motor neurons and interneurons. Theseneurons control movement of the GI tract, as well as trigger specificcells in the gut to release acid, digestive enzymes, and hormonesincluding gastrin, cholecystokinin and secretin. Syndromes that includeinadequate secretion of any of these cellular products may be treatedwith optogenetic stimulation of the producing cell types, or neuronsthat prompt their activity.

Conversely, optogenetic stabilization may be used to treat syndromes inwhich excessive endocrine and exocrine products are being created.Disorders of lowered intestinal motility, ranging from constipation(particularly in patients with spinal cord injury) to megacolan may betreated with optogenetic excitation of motor neurons in the intestines.

Disorders of intestinal hypermotility, including some forms of irritablebowel syndrome may be treated with optogenetic stabilization of neuronsthat control motility.

Neurogenic gastric outlet obstructions may be treated with optogeneticstabilization of neurons and musculature in the pylons. An alternativeapproach to hypomobility syndromes would be to provide optogeneticexcitation to stretch-sensitive neurons in the walls of the gut,increasing the signal that the gut is full and in need of emptying.

In this same paradigm, an approach to hypermobility syndromes of the gutwould be to provide optogenetic stabilization to stretch receptorneurons in the lower GI, thus providing a “false cue” that the gut wasempty, and not in need of emptying. In the case of frank fecalincontinence, gaining improved control of the internal and externalsphincters may be preferred to slowing the motility of the entire tract.During periods of time during which a patient needs to hold feces in,optogenetic excitation of the internal anal sphincter will provide forretention. Providing optogenetic stimulation to the external sphinctermay be used to provide additional continence. When the patient isrequired to defecate, the internal anal sphincter, and then externalanal sphincter should be relaxed, either by pausing the optogeneticstimulation, or by adding optogenetic stabilization.

Conductive hearing loss may be treated by the use of optical cochlearimplants. Once the cochlea has been prepared for optogeneticstimulation, a cochlear implant that flashes light may be used.Sensorineural hearing loss may be treated through optical stimulation ofdownstream targets in the auditory pathway.

Another embodiment of the present invention is directed toward thetreatment of blood pressure disorders, such as hypertension.Baroreceptors and chemoreceptors in regions such as the aorta (aorticbodies and paraaortic bodies) and the carotid arteries (“caroticbodies”) participate in the regulation of blood pressure and respirationby sending afferents via the vagus nerve (CN X), and other pathways tothe medulla and pons, particularly the solitary tract and nucleus.Optogenetic excitation of the carotid bodies, aortic bodies, paraorticbodies, may be used to send a false message of “hypertension” to thesolitary nucleus and tract, causing it to report that blood pressureshould be decreased. Optogenetic excitation or stabilization directly toappropriate parts of the brainstem may also be used to lower bloodpressure. The opposite modality causes the optogenetic approach to serveas a pressor, raising blood pressure. A similar effect may also beachieved via optogenetic excitation of the Vagus nerve, or byoptogenetic stabilization of sympathetic fibers within spinal nervesT1-T4. In an alternative embodiment, hypertension may be treated withoptogenetic stabilization of the heart, resulting in decreased cardiacoutput and lowered blood pressure. According to another embodiment,optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. In yet anotheralternative embodiment, hypertension may be treated by optogeneticstabilization of vascular smooth muscle. Activating light may be passedtranscutaneousiy to the peripheral vascular bed.

Another example embodiment is directed toward the treatment ofhypothalamic-pituitary-adrenal axis disorders. In the treatment ofhypothyroidism, optogenetic excitation of parvocellular neuroendocrine,neurons in the paraventricular and anterior hypothalamic nuclei can beused to increase secretion of thyrotropin-releasing hormone (TRH). TRH,in turn, stimulates anterior pituitary to secrete TSH. Conversely,hyperthyroidism may be treated with optogenetic stabilization of theprovocellular neuroendocrine neurons. For the treatment of adrenalinsufficiency, or of Addison's disease, optogenetic excitation ofparvocellular neuroendocrine neurons in the supraoptic nucleus andparaventricular nuclei may be used to increase the secretion ofvasopressin, which, with the help of corticotropin-releasing hormone(CRH), stimulate anterior pituitary to secrete ACTH. Cushing syndrome,frequently caused by excessive ACTH secretion, may be treated withoptogenetic stabilization of the parvocellular neuroendocrine neurons ofsupraoptic nucleus via the same physiological chain of effects describedabove. Neuroendocrine neurons of the arcuate nucleus produce dopamine,which inhibits secretion of prolactin from the anterior pituitary.Hyperprolactinemia can therefore be treated via optogenetic excitation,while hypoprolactinemia can be treated with optogenetic stabilization ofthe neuroendocrine cells of the arcuate nucleus.

In the treatment of hyperautonomic states, for example anxietydisorders, optogenetic stabilization of the adrenal medulla may be usedto reduce norepinephrine output. Similarly, optogenetic stimulation ofthe adrenal medulla may be used in persons with need for adrenalinesurges, for example those with severe asthma, or disorders that manifestas chronic sleepiness.

Optogenetic stimulation of the adrenal cortex will cause release ofchemicals including Cortisol, testosterone, and aldosterone. Unlike theadrenal medualla, the adrenal cortex receives its instructions fromneuroendocrine hormones secreted from the pituitary and hypothalamus,the lungs, and the kidneys. Regardless, the adrenal cortex is amenableto optogenetic stimulation. Optogenetic stimulation of thecortisol-producing cells of the adrenal cortex may be used to treatAddison's disease. Optogenetic stabilization of cortisol-producing cellsof the adrenal cortex may be used to treat Cushing's disease.Optogenetic stimulation of testosterone-producing cells may be used totreat disorders of sexual interest in women: Optogenetic stabilizationof those same cells may be used to decrease facial hair in women.Optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. Optogeneticexcitation of aldosterone- producing cells within the adrenal cortex maybe used to increase blood pressure.

Optogenetic excitation stimulation of specific affected brain regionsmay be used to increase processing speed, and stimulate growth andinterconnection of neurons, including spurring the maturation of neuralprogenitor cells. Such uses can be particularly useful for treatment ofmental retardation.

According to another embodiment, various muscle diseases and injuriescan be treated. Palsies related to muscle damage, peripheral nervedamage and to dystrophic diseases can be treated with optogeneticexcitation to cause contraction, and optogenetic stabilization to causerelaxation. This latter relaxation via optogenetic stabilizationapproach can also be used to prevent muscle wasting, maintain tone, andpermit coordinated movement as opposing muscle groups are contracted.Likewise, frank spasticity can be treated via optogenetic stabilization.

In areas as diverse as peripheral nerve truncation, stroke, traumaticbrain injury and spinal cord injury, there is a need to foster thegrowth of new neurons, and assist with their integration into afunctional network with other neurons and with their target tissue.Re-growth of new neuronal tracts may be encouraged via optogeneticexcitation, which serves to signal stem cells to sprout axons anddendrites, and to integrate themselves with the network. Use of anoptogenetic technique (as opposed to electrodes) prevents receipt ofsignals by intact tissue, and serves to ensure that new target tissuegrows by virtue of a communication set up with the developing neurons,and not with an artificial signal like current emanating from anelectrode.

Obesity can be treated with optogenetic excitation to the ventromedialnucleus of the hypothalamus, particularly the pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) of thearcuate nucleus. In an alternative embodiment, obesity can be treatedvia optogenetic stabilization of the lateral nuclei of the hypothalamus.In another embodiment, optogenetic stimulation to leptin-producing cellsor to cells with leptin receptors within the hypothalamus may be used todecrease appetite and hence treat obesity.

Destructive lesions to the anterior capsule and analogous DBS to thatregion are established means of treating severe, intractableobsessive-compulsive disorder 48 (OCD48). Such approaches may beemulated using optogenetic stabilization to the anterior limb of theinternal capsule, or to regions such as BA32 and Cg24 which showmetabolic decrease as OCD remits.

Chronic pain can be treated using another embodiment of the presentdisclosure. Electrical stimulation methods include local peripheralnerve stimulation, local cranial nerve stimulation and “sub threshold”motor cortex stimulation. Reasonable autogenic approaches includeoptogenetic stabilization at local painful sites. Attention to promoterselection would ensure that other sensory and motor fibers would beunaffected.

Selective optogenetic excitation of interneurons at the primary motorcortex also may provide effective pain relief. Also, optogeneticstabilization at the sensory thalamus, (particularly medial thalamicnuclei), periventricular grey matter, and ventral raphe nuclei, may beused to produce pain relief. In an alternative embodiment, optogeneticstabilization of parvalbumin-expressing cells targeting as targetingstrategy, may be used to treat pain by decreasing Substance Pproduction. The release of endogenous opiods may be accomplished byusing optogenetic excitation to increase activity in the nucleusaccumbens. In an alternative embodiment, when POMC neurons of thearcuate nucleus of the medial hypothalamus are optogenetically excited,beta endorphin are increased, providing viable treatment approaches fordepression and for chronic pain.

Certain personality disorders, including the borderline and antisocialtypes, demonstrate focal deficits in brain disorders including“hypofrontality.” Direct or indirect optogenetic excitation of theseregions is anticipated to produce improvement of symptoms. Abnormalbursts of activity in the amygdala are also known to precipitate sudden,unprompted flights into rage: a symptom of borderline personalitydisorder, as well as other conditions, which can benefit fromoptogenetic stabilization of the amygdala. Optogenetic approaches couldimprove communication and synchronization between different parts of thebrain, including amygdala, striatum, and frontal cortex, which couldhelp in reducing impulsiveness and improving insight.

The amygdalocentric model of post-traumatic-stress disorder (PTSD)proposes that it is associated with hyperarousal of the amygdala andinsufficient top-down control by the medial prefrontal cortex and thehippocampus. Accordingly, PTSD may be treated with optogeneticstabilization of the amygdale or hippocampus.

Schizophrenia is characterized by abnormalities including auditoryhallucinations. These might be treated by suppression of the auditorycortex using optogenetic stabilization. Hypofrontality associated withschizophrenia might be treated with optogenetic excitation in theaffected frontal regions. Optogenetic approaches could improvecommunication and synchronization between different parts of the brainwhich could help in reducing misattribution of self-generated stimuli asforeign.

Optogenetic stabilization of cells within the arcuate nucleus of themedial hypothalamus, which contain peptide products ofpro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulatingtranscript (CART), can be used to reduce compulsive sexual behavior.Optogenetic excitation of cells within the arcuate nucleus of the medialhypothalamus which contain peptide products of pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) may beused to increase sexual interest in the treatment of cases of disordersof sexual desire. In the treatment of disorders of hypoactive sexualdesire testosterone production by the testes and the adrenal glands canbe increased through optogenetic excitation of the pituitary gland.Optogenetic excitation of the nucleus accumbens can be used for thetreatment of anorgasmia.

The suprachiasmatic nucleus secretes melatonin, which serves to regulatesleep/wake cycles. Optogenetic excitation to the suprachiasmic nucleuscan be used to increase melatonin production, inducing sleep, andthereby treating insomnia. Orexin (hypocretin) neurons strongly excitenumerous brain nuclei in order to promote wakefulness. Optogeneticexcitation of orexin-producing cell populations can be used to treatnarcolepsy, and chronic daytime sleepiness.

Optogenetic stimulation of the supraoptic nucleus may be used to inducesecretion of oxytocin, can be used to promote parturition duringchildbirth, and can be used to treat disorders of social attachment.

Like muscular palsies, the motor functions that have been de-afferentedby a spinal cord injury may be treated with optogenetic excitation tocause contraction, and optogenetic stabilization to cause relaxation.This latter relaxation via optogenetic stabilization approach may alsobe used to prevent muscle wasting, maintain tone, and permit coordinatedmovement as opposing muscle groups are contracted. Likewise, frankspasticity may be treated via optogenetic stabilization. Re-growth ofnew spinal neuronal tracts may be encouraged via optogenetic excitation,which serves to signal stem cells to sprout axons and dendrites, and tointegrate themselves with the network.

Stroke deficits include personality change, motor deficits, sensorydeficits, cognitive loss, and emotional instability. One strategy forthe treatment of stroke deficits is to provide optogenetic stimulationto brain and body structures that have been deafferented from excitatoryconnections. Similarly, optogenetic stabilization capabilities can beimparted on brain and body structures that have been deafferented frominhibitory connections.

Research indicates that the underlying pathobiology in Tourette'ssyndrome is a phasic dysfunction of dopamine transmission in corticaland subcortical regions, the thalamus, basal ganglia and frontal cortex.In order to provide therapy, affected areas are preferably firstidentified using techniques including functional brain imaging andmagnetoencephalography (MEG). Whether specifically identified or not,optogenetic stabilization of candidate tracts may be used to suppressmotor tics. Post-implantation empirical testing of device parametersreveals which sites of optogenetic stabilization, and which areunnecessary to continue.

In order to selectively excite/inhibit a given population of neurons,for example those involved in the disease state of an illness, severalstrategies can be used to target the optogenetic proteins/molecules tospecific populations.

For various embodiments of the present invention, genetic targeting maybe used to express various optogenetic proteins or molecules. Suchtargeting involves the targeted expression of the optogeneticproteins/molecules via genetic control elements such as promoters (e.g.,Parvalbumin, Somatostatin, Cholecystokinin, GFAP), enhancers/silencers(e.g., Cytomaglovirus Immediate Early Enhancer), and othertranscriptional or translational regulatory elements (e.g., WoodchuckHepatitis Virus Post-transcriptional Regulatory Element). Permutationsof the promoter+enhancer+regulatory element combination can be used torestrict the expression of optogenetic probes to genetically-definedpopulations.

Various embodiments of the present invention may be implemented usingspatial/anatomical targeting. Such targeting takes advantage of the factthat projection patterns of neurons, virus or other reagents carryinggenetic information (DNA plasmids, fragments, etc.), can be focallydelivered to an area where a given population of neurons project to. Thegenetic material will then be transported back to the bodies of theneurons to mediate expression of the optogenetic probes. Alternatively,if it is desired to label cells in a focal region, viruses or geneticmaterial may be focally delivered to the interested region to mediatelocalized expression.

Gene Delivery Systems

Various gene delivery systems are useful in implementing one or moreembodiments of the present disclosure. One such delivery system isAdeno-Associated Virus (AAV). AAV can be used to deliver apromoter+optogenetic probe (opsin) cassette to a specific region ofinterest. As used herein, “optogenetic probe” refers to an opsin, e.g.,an opsin, or a variant opsin, of the present disclosure. The choice ofpromoter will drive expression in a specific population of neurons. Forexample, using the CaMKIIα promoter will drive excitatory neuronspecific expression of optogenetic probes. AAV will mediate long-termexpression of the optogenetic probe (opsin) for at least one year ormore. To achieve more specificity, AAV may be pseudotyped with specificserotypes 1, 2, 3, 4, 5, 6, 7, and 8, with each having different tropismfor different cell types. For instance, serotype 2 and 5 is known tohave good neuron-specific tropism.

Another gene delivery mechanism is the use of a retrovirus. HIV or otherlentivirus-based retroviral vectors may be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.Retroviruses may also be pseudo-typed with the Rabies virus envelopeglycoprotein to achieve retrograde transport for labeling cells based ontheir axonal projection patterns. Retroviruses integrate into the hostcell's genome, therefore are capable of mediating permanent expressionof the optogenetic probes. Non-lentivirus based retroviral vectors canbe used to selectively label dividing cells.

Gutless Adenovirus and Herpes Simplex Virus (HSV) are two DNA-basedviruses that can be used to deliver promoter+optogenetic probe cassetteinto specific regions of the brain as well. HSV and Adenovirus have muchlarger packaging capacities and therefore can accommodate much largerpromoter elements and can also be used to deliver multiple optogeneticprobes or other therapeutic genes along with optogenetic probes.

Focal Electroporation can also be used to transiently transfect neurons.DNA plasmids or fragments can be focally delivered into a specificregion of the brain. By applying mild electrical current, surroundinglocal cells will receive the DNA material and expression of theoptogenetic probes.

In another instance, lipofection can be used by mixing genetic materialwith lipid reagents and then subsequently injected into the brain tomediate transfection of the local cells.

Various embodiments involve the use of various control elements. Inaddition to genetic control elements, other control elements(particularly promoters and enhancers whose activities are sensitive tochemical, magnetic stimulation or infrared radiation) can be used tomediate temporally-controlled expression of the optogenetic probes. Forexample, a promoter whose transcriptional activity is subject toinfrared radiation allows one to use focused radiation to fine tune theexpression of optogenetic probes in a focal region at only the desiredtime.

Parkinson's Disease can be treated by expressing optogeneticstabilization in the glutamatergic neurons in either the subthalamicnucleus (STN) or the globus pallidus interna (GPi) using anexcitatory-specific promoter such as CaMKIIa, and apply optogeneticstabilization. Unlike electrical modulation in which all cell-types areaffected, only glutamatergic STN neurons would be suppressed.

Disease Models

Aspects of the present disclosure provide for testing a model of aneural circuit or disease. The model can define output response of thecircuit as a function of input signals. The output response can beassessed using a number of different measurable characteristics. Forinstance, characteristics can include an electrical response ofdownstream neurons and/or behavioral response of a patient. To test themodel, optogenetic probes are expressed at an input position for themodel. The optogenetic probes are stimulated and the outputcharacteristics are monitored and compared to an output predicted by themodel.

In certain implementations, the use of optogenetic probes allows forfine tuning of models defined using electrical probes. As electricalprobes provide only limited ability to direct the stimulus and thus arenot well suited for stimulus of certain areas without also directlystimulating nearby areas. Optogenetic probes disclosed herein provide amechanism for more precise selection of the stimulus location. Forinstance, the stimulus from the optogenetic probes can be directed tovery specific types of circuits/cells, such as afferent fibers. Thefollowing description provides an example implementation consistent withsuch an embodiment and is meant to show the feasibility and wide-rangingapplicability for aspects of present invention.

According to one embodiment of the present disclosure, the invention maybe used in animal models of DBS, for example in Parkinsonian rats, toidentify the target cell types responsible for therapeutic effects (anarea of intense debate and immense clinical importance). This knowledgealone may lead to the development of improved pharmacological andsurgical strategies for treating human disease.

One such application involves long-term potentiation (LTP) and/orlong-term depression (LTD) between two neural groups. By targeting theexpression of a subject opsin to different neural populations andstimulating each with a different frequency of light, LTP or LTD can beaccomplished between the two groups. Each group can be individuallycontrolled using the respective wavelength of light. This can beparticularly useful for applications in which the spatial arrangement ofthe two groups presents issues with individual control using the samewavelength of light. Thus, the light delivery device(s) are lesssusceptible to exciting the wrong neural group and can be less reliantupon precise spatial location of the optical stimulus.

The delivery of the proteins to cells in vivo can be accomplished usinga number of different deliver devices, methods and systems. On suchdelivery device is an implantable device that delivers a nucleotidesequence for modifying cells in vivo, such as a viral-vector. Theimplantable device can also include a light delivery mechanism. Thelight delivery can be accomplished using, for example, light-emittingdiodes (LEDs), fiber optics and/or Lasers.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Hyperpolarizing Opsins

Material and Methods

All experiments were conducted under protocols approved by the StanfordAdministrative Panel on Laboratory Animal Care.

Molecular Cloning

Lentiviral constructs contained BamHI between the promoter and theopsin, NotI between the opsin and the fluorophore, and EcoRI between thefluorophore and the WPRE. Opsin-eYFP fragments were polymerase chainreaction (PCR)-amplified to add AscI and NheI, usinggtggcgcgccctattacttgtacagctcgtccatg (SEQ ID NO:11) (for all opsins),tatgctagccaccatggactatggcggcgc (SEQ ID NO: 12) (for the ChR2 mutants),and gttatgctagcgccaccatgtcgcggaggccatggc (SEQ ID NO:13) (for ChIEF), andthen ligated to an AAV-Ef1α-DIO backbone cut with those sites.

Mac and Arch were obtained from Addgene as green fluorescent protein(GFP) fusion genes, and switched to enhanced yellow fluorescent protein(eYFP) for consistency. Humanized ArchT was synthesized by DNA2.0. Mac,Arch, and ArchT were enhanced to the 2.0 versions using the endoplasmicreticulum (ER) export element alone and to the 3.0 versions with boththe ER export motif and the trafficking signal as describedpreviously³³.

All constructs were fully sequenced to check for accuracy and all AAVvectors were tested for in vitro expression prior to viral production.Complete sequence information is on the website:www(dot)optogenetics(dot)org.

Hippocampal Neuron Culture and Calcium Phosphate Transfections

Primary cultured hippocampal neurons were prepared from P0Sprague-Dawley rat pups (Charles River). CA1 and CA3 were isolated,digested with 0.4 mg/mL papain (Worthington), and plated onto glasscoverslips pre-coated with 1:30 Matrigel (Beckton Dickinson Labware).Cultures were maintained in a 5% CO₂ humid incubator with Neurobasal-Amedium (Invitrogen) containing 1.25% fetal bovine serum (FBS) (Hyclone),4% B-27 supplement (Gibco), 2 mM Glutamax (Gibco), and 2 mg/mL5-Fluoro-2′-deoxyuridine (FUDR) (Sigma), and grown on coverslips in a24-well plate at a density of 65,000 cells per well.

For each well a DNA/CaCl₂ mix was prepared with 2 μg DNA (Qiagenendotoxin-free preparation) and 1.875 μL 2M CaCl₂ (final Ca²⁺concentration 250 mM) in 15 μL H₂O. To DNA/CaCl₂ was added 15 μL of 2×HEPES-buffered saline (pH 7.05). After 20 mM at room temperature (RT),the mix was added drop-wise into each well (from which the growth mediumhad been removed and replaced with pre-warmed MEM) and transfectionproceeded for 45-60 minutes at 37° C., after which each well was washedwith 3×1 mL warm MEM before the original growth medium was returned.

Stereotactic Injections

Adeno-associated virus (AAV) serotype 2/5 was produced by the Universityof Carolina Chapel Hill Vector Core. Genomic titers were 1.5×10¹² cfumL⁻¹ for ChETA_(A), ChETA_(TR), and ChIEF, and 4×10¹² cfu mL⁻¹ for eYFP,eNpHR3.0, and eArch3.0. 1 μL of virus was stereotactically injectedbilaterally into the medial prefrontal cortex of 3-4 week-old mice at+1.7 anteroposterior, 0.4 mediolateral, and 2.5 dorsoventral (in mm frombregma).

Whole-Cell Electrophysiology Recordings

Recordings in cultured neurons were performed 4-6 days post-transfectionin Tyrode's solution (320 mOsm): 125 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 2 mMMgCl₂, 30 mM glucose, and 25 mM HEPES, titrated to pH 7.3-7.4 with NaOH.Tyrode was perfused at a rate of 1-2 mL min⁻¹ and was kept at roomtemperature (20-22° C.). Intracellular solution (300 mOsm) contained 130mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, and 2 mM MgCl₂,titrated to pH 7.3 with KOH. Characterization of excitatory opsins wasdone with bath-applied tetrodotoxin (TTX) (1 μM; Sigma-Aldrich) andintracellular QX-314 chloride (1 mM; Tocris Bioscience). In vitropatching of hyperpolarizing opsins and current clamp recordings fordepolarizing opsins were performed in the presence of synaptictransmission blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM;Sigma-Aldrich) and D(−)-2-amino-5-phosphonovaleric acid (APV; 25 μM,Sigma-Aldrich) as well as gabazine for the current clamp experiments (10μM; Sigma-Aldrich). All recordings of cultured neurons were performed onan upright Leica DM-LFSA microscope.

Recordings of eYFP, eNpHR3.0, and eArch3.0-expressing pyramidal cellswere performed in acute slices from wild-type C57BL/6 mice 6-7 weeksafter virus injection. ACSF contained CNQX, APV, and gabazine.Intracellular solution (280 mOsm) contained 135 mM K-gluconate, 5 mMKCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM MgCl₂, 2 mM Mg-ATP, and 0.2 mMNa₂-GTP, titrated to pH 7.4 with KOH. Pyramidal cells were identified bymorphology and characteristic electrophysiological properties.Recordings were performed on an upright Olympus BX51 microscope. For allpatching experiments, borosilicate glass (Sutter Instruments) pipetteresistances were 3-6 MΩ. For cell-attached electrophysiology recordings,upon obtaining GΩ seals, holding potential was set so that no netcurrent flowed across the membrane; the same stimulation protocol usedfor whole-cell spiking experiments. After the cell-attached recordinghad been performed, we applied suction to the pipette to break into thecell and repeated the same experiments in whole-cell to provide a directwithin-cell comparison. No exogenous retinal co-factor was added toneurons in any preparation.

Light delivery

All experiments were performed using single-photon activation. Forcultured neurons, light was emitted from a 300 W DG-4 lamp (SutterInstruments, Novato, Calif.) and was delivered through a 40×, 0.8 NAwater-immersion objective. Pulsed input signals were delivered to theDG-4 from pClamp (Axon Instruments) via a BNC connection. The delay fromthe DG-4 trigger signal to full light output was measured using anamplified photodetector (Thorlabs) as ˜1 ms, with a 200 μs rise-time.All measurements of time-to-peak and latency were corrected for thisdelay.

For light sensitivity measurements, light was passed through a 470/40 nmfilter (for blue-light sensitive excitatory opsins) or a 562/40 nmfilter (for C1V1s and all inhibitory opsins), and then through a seriesof neutral-density (ND) filters to achieve power densities ranging from˜0.1 to 20 mW mm⁻². Other properties were studied at ˜5 mW mm⁻². Forthese experiments, the light was passed through a Lambda 10-3 filterwheel (Sutter Instruments) with a 10-position wheel for filters ofdifferent wavelengths, ND-normalized to generate closely-matched powerdensities. Filters were: 406/15; 427/20; 445/20; 470/20; 494/20; 520/15;542/20; 560/25; 590/20. Inhibitory spectra also used a 607/45 filter.Functional performance of depolarizing tools in culture used a 470/40 nmfilter (for blue-light sensitive excitatory tools) or a 562/40 nm filter(for C1VTs), and then ND filters to achieve power densities of 2, 6, and20 mW mm⁻².

For experiments investigating fast depolarizing tools in slice, lightwas emitted from the same 300 W DG-4 lamp (Sutter Instruments) anddelivered through a 40×, 0.8NA water-immersion objective. Light waspassed through a 470/40 nm filter and adjusted to achieve a light powerdensity of 5.1 mW mm⁻². For experiments investigating hyperpolarizingtools in slice, a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus), XCitehalogen light source (EXPO) was used. Light was passed through a 589/15filter (eNpHR3.0) or a 560/14 filter (eArch3.0). For experimentscomparing the photocurrent and hyperpolarization magnitudes undermatched conditions, light power density was adjusted to ˜5 mW mm⁻². Forthe remaining experiments light was adjusted across a range of lightpower densities (5-10 mW mm⁻² for eNpHR3.0; 0.25-5 mW mm⁻² for eArch3.0)in order to achieve a comparable range of photocurrents for both opsins.

All experiments contained at least 30 s of dark between sweeps in orderto allow recovery to baseline. All filters are given here as wavelengthin nm/bandwidth in nm. All light power densities were measured comingout of the 40× objective, at approximately the sample distance.

Data Analysis

Analyses of physiological results were performed using ClampFit software

(Axon Instruments) or custom software written in MATLAB (Mathworks).

Access resistance (R_(a)) and input resistance (R_(in)) were monitoredcontinually and data was only included when R_(a) was <30 MΩ and R_(in)was >90 MΩ. Any traces containing escaped spikes were excluded fromanalyses of peak photocurrent or of kinetics, but steady-statephotocurrents were still measured when possible. For current clamprecordings in culture, only cells that fit those criteria and had leakcurrents >−150 pA (holding at −65 mV) were included for analysis. Forcurrent clamp recordings in acute slice, only cells that fit thosecriteria and had resting potentials <−55 mV were included for analysis.

To identify the peak photocurrent, traces were smoothed using the robustLoess method with a filter width of 2 ms and the peak was defined as theextremum from laser onset to 200 ms post laser onset, less the baselinecurrent (from the average over 500 ms prior to laser onset). Visualinspection ensured that no escape spikes or other anomalies occurred.Time-to-peak was measured from laser onset to this marked peak time. Thesteady-state photocurrent was determined by fitting a monoexponentialcurve to the smoothed waveform from 2 ms after the peak to the laseroffset time. Steady-state current was taken from the parameters of thisfit. τ_(off) and τ_(des) were calculated using ClampFit. The trace wasfirst smoothed using a lowpass Gaussian filter with a −3 dB cutoff at1,000 Hz; then a monoexponential curve was fit to the smoothed waveform.All curves were visually inspected for goodness of fit.

Photocurrent properties of the depolarizing tools ChR2, ChETA_(A), andChIEF were characterized in vitro using both the lentiviral and theadenoassociated virus (AAV) constructs. For parameters that depend onsingle-molecule properties (steady-state: peak ratio, action spectrum,light sensitivity, and kinetics), values were pooled across experimentsafter confirming that datasets were not statistically different.Photocurrent properties of the hyperpolarizing tools were assessed intwo separate rounds of experiments. eNpHR3.0 photocurrent magnitudeswere statistically different between the two datasets, so we onlycombine datasets when considering normalized values, or intrinsicsingle-molecule properties (action spectrum, light sensitivity, andkinetics) after confirming that eNpHR3.0 performed similarly acrossdatasets.

Whole-cell spikes were defined as rising above a high threshold (−20 mVfor the comparison of fast depolarizing opsins in slice; 0 mV for allother comparisons) and then dropping below a low threshold (−30 mV).Subsequent spikes that occurred within 2 ms of a prior spike wereignored. To detect spikes elicited by light, a window of time from 1-50ms after the pulse onset was defined. Above 20 Hz, this window wastruncated to 1 ms after the current pulse onset to 1 ms after thesubsequent pulse onset. The window around the last light pulse wastruncated to the same length. Cell-attached spikes were identified usingthe threshold function in ClampFit. Very small, broad events were notincluded as spikes. Where the spike data was ambiguous, the trace wasinspected manually. For each whole-cell pulse train we calculated theproportion of light pulses that elicited ≥1 spike (pulse efficacy) andthat elicited >1 spike (multiple spike likelihood).

Plateau potentials were defined as the offset of the spike waveform fromthe baseline. For the depolarizing tools in vitro, all cells that fired≥one spike were included for analysis. For the fast-spiking cells inslice, only traces that had 100% pulse efficacy were included foranalysis. Temporal stationarity, the extent to which spiking issustained at the same reliability over time, was calculated by dividingthe light pulses into quartiles and computing the pulse efficacy eachquartile. Latency and latency spread across pulse trains were determinedas follows: For each light pulse, we measured the time delta from thelight pulse onset to the spike time. Latency is the average of thesetime deltas, and latency spread is the standard deviation of these timedeltas. Note that latency spread therefore is a measure of how variablethe latencies are within each cell, whereas the error bars on latencyare the standard error of mean latencies across cells. Traces in whichthe cell fired <5 action potentials were excluded from analysis.

Statistical Analysis

All statistical analysis was performed using Graphpad Prism version 5.04for Windows (GraphPad Software, www(dot)graphpad(dot)com). Fortwo-sample comparisons of a single variable (such as kinetics ofChETA_(A) vs. ChIEF in slice) it was first tested whether the datafollowed a Gaussian distribution (Shapiro-Wilk normality test). If thedata were detectably non-Gaussian, a non-parametric Mann-Whitney testwas performed. If the data well-approximated a Gaussian, an independent,two-sample t-test (equal variance) was performed. In the case of unequalvariance (determined by an F test), Welch's correction was applied. Alltests were two-tailed with confidence levels of 95%.

For multi-way comparisons of a single variable (such as kinetics of alldepolarizing opsins in culture) it was first tested whether the datafollowed a Gaussian distribution (Shapiro-Wilk normality test). In casesin which distributions were detectably non-Gaussian, a square roottransformation was used to stabilize the variance and make the dataapproximately normal; all data were then compared against one specified“control”, correcting for family-wise error using Dunnett's test. If thetransformed data were still non-Gaussian, we used the non-parametricDunn's test. In all cases, overall significance levels of alpha=0.05(95% confidence interval) were maintained. Comparisons between largernumbers of opsins will therefore have a more conservative alpha (morestringent requirement for significance). This may also result indifferent significance values assigned to the same comparison, dependingon how many comparisons are being performed in parallel. In particular,since some of the same ChR2 and ChETA_(A) data were included in twocomparisons, discrepancies in reported significance values can beattributed to the total number of opsins included in each set ofcomparisons.

For comparisons across multiple variables (such as spiking performanceacross frequencies), two-way ANOVAs were performed, followed bypost-tests between pairs or against a specified “control”. Aconservative Bonferroni's correction was used to control the falsepositive rate. To test the relationship between two opsin properties(such as τ_(off) vs. EPD50), a nonparametric, two-tailed Spearmancorrelation with a confidence level of 95% was performed. To estimatethe slope, a least-squares regression (either linear or linear onlog-log transformed data), minimizing relative distance squared (1/Y^2)was performed.

To test the dependency of an opsin property on an experimental condition(e.g. photocurrent vs. light power density), regressions were performed,as follows. First, for analysis of time-to-peak vs. light power density,we performed linear regression on log-log transformed data wasperformed; and it was compared whether, for each opsin, the best-fitslope differs significantly from 0. Second, for analysis of recoveryfrom desensitization, a non-linear regression was used to fit the meanphotocurrent recovery data with a two-phase association curve,constraining Y₀=0 and plateau =1. This fit was used to generate thecurves and the R-squared values. In a separate analysis, we fit the datafor each individual cell, to calculate the time required for 50%recovery. Third, for analysis of light sensitivities, the raw populationmeans was fit with a one-site specific binding curve:Y=B_(max)*X/(Kd+X). In a separate analysis, the photocurrents for eachcell were normalized; and the population means and standard errors foreach opsin were plotted. This population data was fit the same way togenerate the curves and the R-squared values. For each individual cell,a Kd (equilibrium binding constant), which we refer to as EPD50 (50%effective light power density), was obtained.

Population significance thresholds were always set at P<0.05 (*), P<0.01(**), and P<0.001 (***) for the entire family of comparisons. All graphsare shown as mean±standard error of the mean (s.e.m.).

Immunohistochemistry

6 or 4 weeks post-injection, mice were perfused transcardially with PBSfollowed by 4% paraformaldehyde (PFA). After an overnight post-fix inPFA, brains were equilibrated in 30% sucrose in PBS for at least 24hours. 40 μm sections were obtained using a frozen microtome,DAPI-stained (1:50,000), and coverslipped with PVA-DABCO(Sigma-Aldrich). Transfected primary hippocampal cultures were fixed for15 min with 4% PFA. For staining with KDEL (SEQ ID NO: 14), cultureswere then permeabilized for 30 min with 0.4% saponin in 2% normal donkeyserum (NDS). Primary antibody incubations were performed overnight at 4°C. using a monoclonal antibody marking endogenous ER-resident proteinscontaining the KDEL (SEQ ID NO:14) retention signal (anti-KDEL 1:200,Abcam). Secondary antibodies (Jackson Laboratories) were applied in 2%NDS for 1 hour at room temperature.

Equipment and Settings

All images were obtained on a Leica confocal microscope (DM600B) as1024×1024 resolution (pixel dimensions=3.03 μm²). Images were acquiredusing the following objectives: 10×/0.40 NA (air), 40×/1.25 NA (oil),and 63×/1.4 NA (oil). Excitation and emission wavelengths were asfollows: eYFP in FIG. 1b , 514 nm/512-600 nm; eYFP for all otherfigures, 488 nm/500-545 nm; GFP, 488 nm/500-600 nm; Cy5, 633 nm/650-750nm. The following figures used line-averaging: FIG. 1b and FIG. 2a .Consistent settings were used for all images in each given figure panel.The brightness and contrast of all eYFP images for FIG. 1b wereuniformly and identically modified in Photoshop (Adobe). All otherimages were unprocessed after acquisition.

Quantification of Fluorescence Levels in Transfected Cells

Fluorescence images were acquired from the same cells that were patchedto enable quantification of expression levels andphotocurrent/fluorescence relationships. Images were acquired withMetamorph, maintaining constant settings, and processed off-line usingImageJ. Hand-drawn ROIs encompassed the soma and proximal dendrites.

Results

Hyperpolarizing Tools and Properties

Various hyperpolarizing optogenetic tools were compared head-to-head.Although each experiment will have its own unique set of requirementsfor hyperpolarizing photocurrent properties, some common guidingprinciples initially seem clear. First, in most experimentalapplications, hyperpolarizing photocurrents will need to be sufficientlylarge to robustly and safely inhibit spiking even in the presence ofexcitatory inputs. Second, higher light sensitivity will likely enablemodulation of larger volumes of tissue, the use of lower light powers,and/or less invasive light delivery. Third, precise, time-lockedinhibition will presumably require photocurrents with rapid onset andoffset, while longer-term inhibition will require photocurrents that arestable, with minimal desensitization. Finally the nature of the actionspectrum will dictate feasibility of combining with otherlight-activated reagents in the same preparation^(32, 33).

The first hyperpolarizing tool shown to be efficacious in neurons wasthe N. pharaonis halorhodopsin (NpHR), a yellow light-activated chloridepump that has now been used in preparations ranging across mammalianbrain slice³², freely moving worms³², cultured neurons^(32, 34), andbehaving mammals³⁵⁻³⁸. Two versions modified for enhanced membranetargeting in mammalian neurons, termed eNpHR2.0³⁹ and eNpHR3.0³³ havesince been reported. The outward proton pumps Arch⁴⁰ (from Halorubrumsodomense), ArchT⁴¹ (from Halorubrum strain TP009), eBR³³ (fromHalobacterium) and Mac⁴⁰ (from Leptosphaeria maculans) have alsorecently been shown to achieve successful neuronal inhibition. eNpHR3.0has larger photocurrents than eNpHR2.0, and Arch has largerphotocurrents than eNpHR2.0⁴⁰, but no direct comparison between eNpHR3.0and Arch or any of the proton pumps has yet been reported. Below ispresented a direct comparison of the most potent hyperpolarizing opsins(FIG. 1a ), including novel membrane trafficking-enhanced versions ofproton pumps resulting in the highest expression levels and inhibitoryphotocurrents yet described. Properties were charaterized in vitro; thenthe functional performance of two of the most promising candidates inacute slice was tested.

Each hyperpolarizing tool was fused in-frame with enhanced yellowfluorescence protein (eYFP), cloned the opsins into an identicallentiviral backbone with the excitatory CaMKIIα promoter (FIG. 1a ); andthe opsins were expressed in cultured neurons (FIG. 1b ). eNpHR3.0 waswell-targeted to the membrane, but Arch, ArchT, and Mac all showedintracellular accumulations reminiscent of the endoplasmic reticulum(ER)-aggregations observed with NpHR1.0³⁹. The same accumulations werealso observed in the GFP versions of the constructs; the GFP and YFP 1.0versions had similar photocurrents. ER-aggregation was confirmed byco-staining with the ER marker KDEL (SEQ ID NO: 14) (FIG. 1b ).Trafficking modifications applied to eNpHR3.0 were applied to Arch,ArchT, and Mac. These novel trafficking-enhanced versions, which aretermed (by analogy with NpHR version progression) eArch3.0, eArchT3.0,and eMac3.0, had markedly reduced intracellular labeling and improvedmembrane-localization with labeling of cellular processes (FIG. 1b ).Intermediate “2.0” versions were potent but not as successful as the 3.0versions.

Because only those proteins expressed on the membrane can contribute tothe measured photocurrent, it was anticipated that this improved opsintrafficking should increase photocurrent size. Indeed, all threeenhanced proton pumps had dramatically increased photocurrents (P<0.001;FIG. 1c ). While the 1.0 versions of the proton pumps had significantlysmaller photocurrents than eNpHR3.0, eArch3.0 and eArchT3.0photocurrents were significantly larger (P<0.001 for each comparison;FIG. 1c ). eNpHR3.0-expressing cells had the dimmest fluorescence, butthe greatest photocurrent per fluorescence, of these tools.

Although maximal eMac3.0 photocurrents were the smallest among theenhanced opsins (and significantly smaller than eNpHR3.0; P<0.05), Machas been reported to have an activation spectrum sufficientlyblue-shifted to allow dual-inhibition in combination with eNpHR3.0⁶⁴.After verifying that membrane trafficking did not change the spectra,the spectra of the enhanced pumps were compared, and plotted with ChR2,for reference (FIG. 1d ). eNpHR3.0 was red-shifted (peaking at 560-590nm) relative to the three proton pumps (peaking at 520-560 nm),exhibiting the least overlap with ChR2; no functionally relevantdifferences were seen among the proton pumps.

The temporal precision of hyperpolarizing photocurrents was investigatedby quantifying on-kinetics (τ_(on)) and off-kinetics (τ_(off)) at thebeginning and end of a 1 s light pulse. All pumps activated rapidly,with proton pumps activating significantly faster than eNpHR3.0 (allwithin the range of 1.5-3 ms, FIG. 1e ). Both Mac variants had muchslower off-kinetics compared with the other pumps (P<0.001; FIG. 1e ).

The light sensitivity of the hyperpolarizing pumps was assessed bymeasuring photocurrents across a range of light power densities rangingfrom ˜0.05 to ˜20 mW mm⁻² (FIG. 1f ); due to small photocurrents, Mac1.0was eliminated from this and subsequent analyses.) As expected, the 3.0pumps had much larger operational light sensitivity (that is, byabsolute current magnitude) than the 1.0 counterparts, althoughtrafficking-enhancement did not affect the population sensitivity(normalized current magnitudes or EPD50). eMac3.0 was the most sensitive(EPD50 =1.9±0.4 mW mm⁻² vs. 5.4±0.2 mW mm⁻² for eNpHR3.0; P<0.001).Off-kinetics and population light sensitivity were therefore inverselycorrelated for the hyperpolarizing tools, reminiscent of the patternobserved for depolarizing tools.

Given that many behavioral neuroscience experiments may requireprolonged inhibition on the order of minutes, the stability of thehyperpolarizing photocurrents was investigated. While all pumpphotocurrents decayed across 60 s of continuous light, eNpHR3.0 currentswere the most persistent and the large 3.0 proton pump currents(eArch3.0 and eArchT3.0) had the largest drop-off in vitro. All pumpsrecovered photocurrents with similar efficacy under thesecultured-neuron conditions.

FIG. 1: Properties of hyperpolarizing tools. (a) NpHR is an inwardchloride pump (halorhodopsin type; HR), while Arch, ArchT, and Mac areoutward proton pumps (bacteriorhodopsin type; BR). 3.0 versions includea trafficking sequence (TS) between opsin and fluorophore and the2.0-type endoplasmic reticulum export sequence (ER) after thefluorophore. (b) Confocal images of 1.0 and 3.0 versions (green)expressed in culture and immunolabeled with an ER marker (anti-KDEL (SEQID NO: 14); red). Horizontal scale bar represents 25 μm. (c)Representative traces and raw photocurrents in response to 1 s light for1.0 (open bars) vs. 3.0 versions (closed bars) for Arch (n=15-19), ArchT(n=14-16), and Mac (n=8-12). Vertical and horizontal scale barsrepresent 500 pA and 500 ms, respectively. Photocurrents were normalizedto eNpHR3.0 values from within the same experiment to enable directcomparisons across opsins (n=835). (d) Action spectra for 3.0 versions(n=7-20) alongside ChR2 (black). (e) τ_(on) and τ_(off) (n=7-35).Vertical and horizontal scale bars represent 200 pA and 5 ms,respectively. (f) EPD50 for all hyperpolarizing opsins (n=5-14). Rawphotocurrent vs. light power density plotted alongside within-experimenteNpHR3.0 (n=5-14). All population data is plotted as mean±s.e.m. Starsindicate significance level: * P<0.05, ** P<0.01, * ** P<0.001. Unlessotherwise indicated, eNpHR3.0 was activated with 590 nm light, while allother tools were activated with 560 nm light, both at ˜5 mW mm⁻².

Hyperpolarizing Tools: Inhibiting Spikes in in Acute Slice

To further investigate the characteristics of prolonged photocurrentsunder conditions more relevant to in vivo experiments, and to test thefunctional ability of hyperpolarization to stably inhibit spiking, acuteslice preparations were used. For this analysis, one of each broad classof hyperpolarizing tool (namely, the chloride pump eNpHR3.0 against oneof the proton pumps) was compared. The enhanced counterpart of thebest-established proton pump (Arch1.0) to date, namely eArch3.0 wasused. To express eNpHR3.0 and eArch3.0 in vivo, an adeno-associatedviral vector (AAV serotype 2/5), with the opsin-eYFP fusion gene undercontrol of the CaMKIIα promoter, was stereotactically injected. Undermatched conditions, eArch3.0 expressed much more strongly based onfluorescence, both at the injection site and in axons at downstreamtargets such as the basolateral amygdala (BLA; FIG. 2a ). Compared witheYFP-transduced controls, cells expressing both opsins had similarbaseline input resistances (FIG. 2b ) and resting potentials, butslightly higher membrane capacitance, as has previously been observedfor opsin-expressing HEK cells⁴². Also as expected from the in vitrowork (FIG. 1), at matched light power densities (5 mW mm⁻²) eArch3.0 hadsignificantly larger photocurrents (P=0.01), averaging 1680±360 pA vs.450±70 pA for eNpHR3.0 (FIG. 2c ). Under current-clamp,eArch3.0-mediated hyperpolarization was also significantly larger(−94±12 mV vs. −41±4 mV, P=0.005; FIG. 2d ); smaller differences inhyperpolarization compared with photocurrent could be due tovoltage-dependent slowing of photocycle turnover in proton pumps.

Because photocurrent stability and cell responses to hyperpolarizationmay depend on photocurrent magnitudes, a set of experiments was carriedusing non-matched light power densities (5-10 mW mm⁻²for eNpHR3.0;0.25-5 mW mm⁻² for eArch3.0) to obtain a similar range of photocurrentsfor the two tools. Cells were illuminated for 60 s under voltage clamp,and measured the start and end photocurrent for each cell. These datawere well-fit by linear regression (eNpHR3.0 R²=0.68, eArch3.0 R²=0.88)with eArch3.0 having significantly higher slope (F_(1,36)=22.2,P<0.001), reflecting the fact that, for cells with similar onsetphotocurrents, eArch3.0-expressing cells had more photocurrent remainingat the end of the light pulse under these slice conditions, as seen inthe illustrative traces and in contrast with the pattern of stabilityobserved in vitro.

The ability of eArch3.0 and eNpHR3.0 to inhibit spiking in current clampwas assessed. Spiking was elicited with modestly suprathreshold currentinjections at 5 Hz, with 30 s baseline (pre-light), 60 s light, and 30 spost-light. Both pumps successfully blocked spikes throughout theduration of the prolonged light stimulation (FIG. 2e ). We observed thatfrom both groups some cells became unstable after prolongedhyperpolarization especially by >50 mV, failing to spike to currentinjections or rebounding to a more depolarized resting potential afterlight offset. These factors were quantified for each cell and plottedeach against the degree of hyperpolarization (FIG. 20. Under moremoderate (>50 mV) hyperpolarizations, no consistent or lasting effectson excitability or membrane resistance were observed.

FIG. 2: Performance of hyperpolarizing tools. (a) Confocal images ofeNpHR3.0 and eArch3.0 expression at the injection site in medialprefrontal cortex (mPFC) and the downstream basolateral amygdala (BLA).Scale bars represent 250 μm and 25 μm. DAPI staining (white) delineatescell bodies. (b) Mean input resistances for opsin-expressing cells andeYFP-controls (n=10-22). (c) Representative traces and mean onsetphotocurrents for eArch3.0 and eNpHR3.0 in response to 60 s 5 mW mm⁻²light pulses (n=8-10). Vertical and horizontal scale bars represent 400pA and 10 s, respectively. (d) Mean peak hyperpolarization generated byeArch3.0 and eNpHR3.0 with 60 s 5 mW mm⁻² light pulses (n=6-10). (e)Suppression of current injection-evoked spiking in reliably-firing cellsby 60 s of continuous light in cells expressing eNpHR3.0 or eArch3.0.Cells were illuminated with light power densities set to achieveapproximately matched hyperpolarization. Vertical and horizontal scalebars represent 40 mV and 20 s, respectively. (f) Relationship betweenhyperpolarization magnitude and cell stability. Post-light recovery ofevoked spiking (relative to pre-light performance) and change in restingpotential plotted against light-evoked hyperpolarization. All populationdata is plotted as mean±s.e.m. Stars indicate significance level: *P<0.05, ** P<0.01, *** P<0.001. eNpHR3.0 was activated with 590 nmlight, while eArch3.0 was activated with 560 nm light.

Example 2 Cloning and Characterization of Dunaliella salina Opsin

Typically found in hyper-saline environments such as evaporation saltfields, the unicellular (oval with two flagella) green alga Dunaliellasalina is salt tolerant. Despite belonging to the same order as thegreen algae Chlamydomonas reinhardtii and Volvox carteri, Dunaliella canappear reddish due to the accumulation of high levels of carotenoidmolecules (FIG. 3A). We hypothesized that a Dunaliella ChR might haveunusual properties and engaged in efforts to clone ChRs from thisflagellated algal species.

Despite high homology with other known ChRs, the DChR1 sequencecontained several notable features (FIG. 3B). First, one of the residuesthat is thought to contribute to the complex counterion of the RSB, E123in ChR2 as discussed above, is replaced by Ala in the DChR1 TM3 (FIG.3B,C); from structural modeling (FIG. 3C), it was expected that thecounterion function is assumed by E309 in DChR1, a position that playsonly a minor role in BR (D212) or Anabaena sensory rhodopsin (ASR)(Vogeley et al., 2004). Even more remarkably, DChR1 photocurrents wereexclusively carried by protons, unlike any other known ChR, and werecompletely unaffected by changes in the extracellular cation composition(FIG. 3D). Consequently, the photocurrent was highly sensitive tochanges in the pH environment and completely vanished at high pH (FIG.3E).

Full understanding of structure-function relationships will requirehigh-resolution crystal structures in multiple photocycle states.However, directed mutagenesis studies here demonstrate that DChR1 has adifferent counterion arrangement and ion selectivity compared to otherknown ChRs. The strict H³⁰ selectivity of DChR1 was not mediated by theunusual protonated retinal Schiff base (RSBH) counter ion, assubstitution of A178 with the more typical putative counterion Glu asfound in ChR2 only red-shifted the activation spectrum (FIG. 3F, from475 to 510 nm) with minimal effect on current amplitude or kinetics.Similarly, replacing E309 with Asp caused a slight spectral shift and aslight current increase, whereas replacing the charged E309 by Alarendered the protein almost totally inactive (FIG. 3F).

Given typical electrochemical proton gradients, the DChR1 H⁺ currentdirection is opposite in direction to the H⁺ current generated bybacteriorhodopsin (BR) pump activity; therefore, DChR1 and BR couldenable interventions such as bidirectional control of cellular pH, forexample in manipulating the pH of intracellular compartments(mitochondria and synaptic vesicles). DChR1 therefore defines a novelclass of microbial opsin—a light-activated proton channel—unlike anyother microbial opsin including ChR1 and ChR2. These findings illustratethe diversity of function likely to be present within the vast array ofmicrobial opsin genomes.

FIG. 3. Characterization of a channelrhodopsin from Dunaliella salina.A. The halophilic unicellular alga Dunaliella salina. B. Sequencehomology between the algal channelrhodopsins and BR within the thirdtransmembrane helix. The typically conserved E123 position has beenreplaced with an Ala in DChR1 (and is shown on a yellow background),conserved residues are shown on a blue background, and amino acidslikely interacting with the chromophore are shown in red. C. Lack of aproton acceptor in DChR1, compared with BR and Chlamydomonas ChR2(CChR2). ASR (Anabaena sensory rhodopsin) has been crystallized with amixture of all-trans retinal seen as an overlay (Vogeley et al., 2004).D. DChR1 photocurrents are unaffected by changes in the extracellularcation composition (sole cation present in each condition shown oncategory X axis). Cation exchange was performed in 5 mM Mops-NMG, 0.1 mMMgCl₂ with 100 mM LiCl, KCl, NaCl, Guanidium chloride or NMG chloride(pH 7.5). We used a human-codon adapted DChR sequence (amino acidresidues 1-339) as a template for capped RNA synthesis by T7 RNApolymerase (mMessage mMachine, Ambion). Oocyte preparation, injection ofcapped RNA were carried out as described previously (Berthold et al.2008), and two-electrode voltage clamp was performed with a Turbo Tec-05(NPI Electronic) or a GeneClamp 500 (Molecular Devices) amplifier on anoocyte after 3-7 days of the capped RNA injection. Continuous light wasprovided by a 75-W Xenon lamp (Jena Instruments) and delivered to theoocytes via a 3-mm light guide. The light passed through a 500 25-nmbroadband filter (Balzers) with an intensity of 46 mW/cm². E. Incontrast, DChR1 photocurrent is highly sensitive to changes in the pHenvironment. Solutions contained 100 mM NMG-chloride, 0.1 mM MgCl₂, 0.1mM CaCl₂ with 5 mM glycine (pH 9.0), 5 mM Mops-NMG (pH 7.5), 5 mMcitrate (pH 6, 5.5, 5.0, 4.6, 4.2). F. Introduction or alteration of aproton acceptor (A178E or E309D) into the DChR1 retinal-binding pocketcauses a pronounced red-shift in the absorption spectrum. We applied10-ns laser flashes as described previously (Berthold et al. 2008);solutions for action spectra recording contained 100 mM NaCl, 0.1 mMMgCl₂, 0.1 mM CaCl₂ and 5 mM citrate (pH 4.2).

A nucleotide sequence endoding D. salina DChR1 is presented in FIG. 4.The DChR1-encoding nucleotide sequence was codon-optimized for mammalianexpression; the codon-optimized nucleotide sequence is depicted in FIG.5. FIG. 6 provides an amino acid sequence of D. salina DChR1.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An isolated fusion polypeptide comprising, inorder from N-terminus to C-terminus: a) an opsin polypeptide comprisingan amino acid sequence having at least 90% amino acid sequence identityto the amino acid sequence set forth in SEQ ID NO:33; b) a membranetrafficking signal comprising the amino acid sequenceKSRITSEGEYIPLDQIDINV (SEQ ID NO:10); and c) an ER export signalcomprising the amino acid sequence FCYENEV (SEQ ID NO:5).
 2. The fusionpolypeptide of claim 1, wherein the opsin polypeptide comprises an aminoacid sequence having at least 95% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:33.
 3. The fusion polypeptideof claim 1, wherein the opsin polypeptide comprises an amino acidsequence having at least 96% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO:33.
 4. The fusion polypeptide ofclaim 1, wherein the opsin polypeptide comprises an amino acid sequencehaving at least 97% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO:33.
 5. The fusion polypeptide of claim1, wherein the opsin polypeptide comprises an amino acid sequence havingat least 98% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:33.